Home

Jump-In™ TI™ Gateway® System

1. The Jump In TI Gateway Vector Kit supplied with the Jump In TI Gateway System is also available individually Cat no A10896 It contains the vectors for retargeting the platform cell line Store the vectors at 20 C Vector Composition Amount pJII R4 DEST 40 ul of vector at 150 ng pl in TE buffer pH 8 0 6 ug pJIT R4 Int 20 ul of vector at 500 ng pl in TE buffer pH 8 0 10 ug Continued on next page Kit Contents and Storage continued MultiSite The following vectors and primers are supplied with the MultiSite Gateway Pro Gateway Pro Plus Plus Vector Module for creating the entry and expression clones in a multi fragment Vector Module recombination reaction Store the contents of the vector module at 20 C Vector Composition Amount pDONR 221 P1 P5r 60 ul of vector at 100 ng pl in TE Buffer pH 8 0 6ug pDONR 221 P5 P2 60 ul of vector at 100 ng pl in TE Buffer pH 8 0 6ug pDONR 221 P1 P4 60 ul of vector at 100 ng pl in TE Buffer pH 8 0 6 ug pDONR 221 P4r P3r 60 ul of vector at 100 ng pl in TE Buffer pH 8 0 6 ug pDONR 221 P3 P2 60 ul of vector at 100 ng pl in TE Buffer pH 8 0 6ug pDONR 221 P5 P4 60 ul of vector at 100 ng pl in TE Buffer pH 8 0 6ug pENTR L1 pLac lacZalpha R5 60 ul of vector at 100 ng pl in TE Buffer pH 8 0 6ug pENTR L5 pLac Spect L2 60 ul of vector at 100 ng pl in TE Buffer pH 8 0 6 ug pENTR
2. Longer incubation times are essential if you are assembling more than two fragments Generally overnight incubation at either room temperature or at 16 C should work Performing multiple transformations is more efficient than performing one large transformation For a 4 fragment assembly it may be necessary to transform the complete reaction volume to get enough colonies for analysis Five transformations of 2 yl each will yield more colonies than two transformations of 5 ul each Replica plate the colonies obtained from transformations on ampicillin and kanamycin plates True recombinant clones will only grow on ampicillin plates Establishing Sensitivity to Selection Agents Introduction Blasticidin Preparing and Storing Blasticidin S Determining Blasticidin S Sensitivity TM After you cotransfect your retargeting expression construct and the pJIT R4 Int vector into your R4 platform cells to create your isogenic cell line you will select stable transformants containing your genetic elements of interest by their resistance to Blasticidin Geneticin G 418 a neomycin analog or Zeocin Successful retargeting of your R4 platform cell line will position the constitutive human EFl1a promoter upstream of the promoterless resistance gene and confer resistance to the appropriate selective agent depending on the pJTI platform vector used To succefully create your isogenic cell line by retargeting you need to
3. When performing PCR analysis on the genomic DNA isolated from your R4 platform line clones you should consider the following factors e We recommend using nested PCR with primary and secondary reactions to eliminate the high background observed with only primary PCR e You should design your primers for the R4 retargeting sequence from the R4 attP site to the appropriate resistance marker Bsd Neo or Zeo depending on the platform vector used You may use the Hygromycin resistance gene from the plasmid DNA as a positive control e For a map and a description of the features of each platform vector pJ IT Bsd pJIT Neo and pJTI Zeo and of the pJTI PhiC31 Int vector see pages 30 33 e The vector sequences of pJTI Bsd pJTI Neo pJTI Zeo and pJTI PhiC31 Int vectors are available on our website at www invitrogen com or by contacting Technical Support see page 42 e We recommend a high fidelity thermostable DNA polymerase such as the AccuPrime Tag DNA Polymerase for the nested PCR see page xi for ordering information e Be sure to include a final extension step 7 minutes at 72 C in your PCR e Follow the protocol below to prepare genomic DNA from crude cell lysates for your PCR Note Other genomic DNA isolation methods are also suitable Pellet a total of 10 000 to 30 000 cells Wash the cells with 500 pl PBS Centrifuge cells to pellet and remove PBS e o ah TES Resuspend the cell pelle
4. TM TM This manual provides an overview of the Jump In TI Gateway System and offers instructions and guidelines for e Generating selecting and expanding your platform cell line using the TM TM Jump In TI Platform Kit e Creating your retargeting expression construct using the MultiSite Gateway Pro Plus Vector Module module and the Jump In TI Gateway Vector Kit e Retargeting your platform line with your retargeting expression construct using the Jump In TI Gateway Vector Kit and the subsequent selection and expansion of your retargeted cell line e Characterization and quality control of your cell line after targeted integration events i e platform line creation and retargeting This manual does not provide detailed protocols for maintaining your mammalian cell culture as each cell line behaves differently under different laboratory conditions However you will find general instructions on maintaining your cells before and after the retargeting events and suggestions and tips on cell culture to ensure successful targeted integration experiments For more information about the MultiSite Gateway Technology refer to the MultiSite Gateway Pro manual 25 0942 supplied with the kit For more information on targeted integration see published literature Thyagarajan et al 2008 Thyagarajan et al 2001 For more information on culturing mammalian cell lines and human stem cells refer to www invitrogen co
5. D MEM 500 ml 11995 065 high glucose with L glutamine and sodium pyruvate D MEM F 12 containing GlutaMAX 1X liquid 500 ml 10565 018 Opti MEM I Reduced Serum Medium 100 ml 31985 062 500 ml 31985 070 OptiPRO SFM 1X 1000 ml 12309 019 CD CHO Medium 1000 ml 10743 029 CD 293 Medium 1000 ml 11913 019 293 SFM II 1000 ml 11686 029 CD DG44 Medium 1000 ml 12610 010 StemPro hESC SFM Complete Medium 1 kit A1000701 contains StemPro supplement D MEM F 12 with GlutaMAX 25 BSA FGF basic and 2 mercaptoethanol Dulbecco s Phosphate Buffered Saline 500 ml 14190 144 D PBS 1X liquid Ca and Mg free 1000 ml 14190 136 10x500ml 14190 250 Dulbecco s Phosphate Buffered Saline 500 ml 14040 133 D PBS 1X liquid contains Ca and Mg 10x500ml 14040 182 Phosphate Buffered Saline PBS pH 7 4 500 ml 10010 023 1000 ml 10010 031 Continued on next page Accessory Products continued Serum and Supplements for Cell Culture Fetal Bovine Serum ES Cell Qualified Mitomycin C Treated MEFs Porcine Skin Gelatin We recommend the following accessory products for culturing passaging and maintaining your mammalian cell and embryonic stem cell cultures For more information on these and other cell culture products available from Invitrogen refer to www invitrogen com or contact Technical Support see page 42 Product Amount Cat no GlutaMAX I Supplement 100 ml 35050 0
6. K and Dower W J 1988 Electroporation of Eukaryotes and Prokaryotes A General Approach to the Introduction of Macromolecules into Cells BioTechniques 6 742 751 Thyagarajan B Liu Y Shin S Lakshmipathy U Scheyhing K Xue H Ellerstrom C Strehl R Hyllner J Rao M S and Chesnut J D 2008 Creation of engineered human embryonic stem cell lines using phiC31 integrase Stem Cells 26 119 126 Thyagarajan B Olivares E C Hollis R P Ginsburg D S and Calos M P 2001 Site specific genomic integration in mammalian cells mediated by phage phiC31 integrase Mol Cell Biol 21 3926 3934 Wigler M Silverstein S Lee L S Pellicer A Cheng Y C and Axel R 1977 Transfer of Purified Herpes Virus Thymidine Kinase Gene to Cultured Mouse Cells Cell 11 223 232 2008 2009 Life Technologies Corporation All rights reserved For research use only Not intended for any animal or human therapeutic or diagnostic use 46 invitrogen Corporate Headquarters Invitrogen Corporation 5791 Van Allen Way Carlsbad CA 92008 T 1 760 603 7200 F 1 760 602 6500 E tech_support invitrogen com For country specific contact information visit our web site at www invitrogen com
7. L1 pLac lacZalpha L4 60 ul of vector at 100 ng pl in TE Buffer pH 8 0 6 ug pENTR R4 pLac Spect R3 60 ul of vector at 100 ng pl in TE Buffer pH 8 0 6ug pENTR L3 pLac Tet L2 60 ul of vector at 100 ng pl in TE Buffer pH 8 0 6 ug pENTR L5 LacI L4 60 ul of vector at 100 ng pl in TE Buffer pH 8 0 6 ug M13 20 Forward primer 20 ul of primer at 100 ng pl in TE Buffer pH 8 0 2 ug M13 Reverse primer 20 ul of primer at 100 ng pl in TE Buffer pH 8 0 2ug pDONR 221 Lyophilized in TE Buffer pH 8 0 6 ug Continued on next page vii Kit Contents and Storage continued LR Clonase II Plus Enzyme Mix BP Clonase II Enzyme Mix One Shot Mach1 T1 Chemically Competent E coli Genotype of Mach1 T1 viii The following reagents are supplied with LR Clonase II Plus enzyme mix Store at 20 C for up to 6 months For long term storage store at 80 C Item Composition Amount LR Clonase II Plus Enzyme Mix Proprietary 40 ul Proteinase K solution 2 ug pul in 40 ul 10 mM Tris HCl pH 7 5 20 mM CaCl 50 glycerol The following reagents are supplied with BP Clonase II enzyme mix Store at 20 C for up to 6 months For long term storage store at 80 C Item Composition Amount BP Clonase II Enzyme Mix Proprietary 40 ul Proteinase K solution 2 ug pl in 40 ul 10 mM Tris HCl pH 7 5 20 mM CaCl 50 glycerol 30 PEG Mg solution 30 PEG 8000 30 mM MgCl 1ml
8. Use correct growth medium bFGF inactive bFGF is not stable when frequently warmed and cooled Add bFGF to medium just before use or store medium with bFGF in aliquots at 20 C Cells too old Use healthy cells under passage 30 do not overgrow Cells too diluted Spin down cells for 4 minutes 200 x g at room temperature aspirate media and dilute cells at higher density Clump size is to small and differentiated Be gentle at time of passage so the clumps of cells don t get too small Mycoplasma Discard cells media and reagents and use early contamination stock of cells with fresh media and reagents Cells differentiated Cells not thawed and Thaw and culture a fresh vial of stem cells Make if using stem cells established on correct medium sure to thaw into the correct medium as recommended by the supplier Suboptimal quality of feeder layer if cells are maintained on feeder layers Check the concentration of feeder cells used Purchase see page x or make see page 34 new batch of mitotically inactivated MEFs if necessary Use Hygromycin resistant MEFs after platform creation Culture conditions not correct Thaw and culture fresh vial of stem cells Follow thawing instructions and subculture maintenance procedures exactly Cells overexposed to collagenase Stem cells are very sensitive to collagenase overexposure Avoid exposing cells to collagenase for more than 3 minut
9. pEXP7 tet 50 ng ul in TE Buffer pH 8 0 20 ul The following reagents are included with the One Shot Mach1 T1 Chemically Competent E coli Store the competent cells at 80 C Reagent Composition Amount Mach1 T1 chemically 21 x 50 ul competent cells S O C Medium 2 Tryptone 6 ml 0 5 Yeast Extract 10 mM NaCl 2 5 mM KCl 10 mM MgCl 10 mM MgSO 20 mM glucose pUC19 Control DNA 10 pg ul in 5 mM Tris HCl 50 ul 0 5 mM EDTA pH 8 0 F 80 IlacZ AM15 AlacX74 hsdR rk m ArecA1398 end A1 ton A Accessory Products The products listed in this section may be used with the Jump In TI Gateway System For accessory products that may be used with the MultiSite Gateway Pro Plus Vector Module refer to the MultiSite Gateway Pro manual 25 0942 supplied with the kit For more information refer to our website at www invitrogen com or contact Technical Support see page 42 Introduction Media and Buffers We recommend the following media and buffers for culturing passaging and for Cell Culture maintaining your mammalian and stem cell cultures For more information on these and other cell culture products available from Invitrogen refer to our website at www invitrogen com or contact Technical Support see page 42 Product Amount Cat no Dulbecco s Modified Eagle Medium D MEM 500 ml 11965 092 Dulbecco s Modified Eagle Medium
10. 1M 20 ml 15603 106 100 ml 15630 080 20 x 100 ml 15630 130 Quant iT dsDNA Assay Kit 0 2 100 ng 1 kit Q 33120 UltraPure Glycogen 100 pl 10814 010 UltraPure Salmon Sperm DNA Solution 5x1ml 15632 011 10 mg ml UltraPure 20X SSC 1000 ml 15557 044 UltraPure 10 SDS Solution 4x 100 ml 15553 027 Water distilled 500 ml 15230 162 Continued on next page Accessory Products continued Selection Agents MultiSite Gateway Pro Kits Competent Cells xii The table below lists ordering information for the selection agents required for TM TM use with the Jump In TI Gateway System Kits Product Amount Cat no Hygromycin B 20 ml 10687 010 Blasticidin 5 HCI 50mg R210 01 Geneticin powder 1g 11811 023 5g 11811 031 25g 11811 098 Geneticin liquid 20 ml 10131 035 100 ml 10131 027 Zeocin 1g R250 01 5g R250 05 Invitrogen offers several MultiSite Gateway Pro kits for rapid construction of expression clones containing your choice of up to four separate DNA elements which allow the opportunity to perform pathway reconstitution multiple gene expression and regulation and protein interaction studies All MultiSite Gateway Pro kits are compatible with the pJTI vectors included in the Jump TM TM In TI Gateway System kits Each kit supplies enough reagents for 20 recombination reactions Product Cat no MultiSite Gateway P
11. Cloning Using in vitro Site Specific Recombination Genome Research 10 1788 1795 Mulsant P Tiraby G Kallerhoff J and Perret J 1988 Phleomycin Resistance as a Dominant Selectable Marker in CHO Cells Somat Cell Mol Genet 14 243 252 Palmer T D Hock R A Osborne W R A and Miller A D 1987 Efficient Retrovirus Mediated Transfer and Expression of a Human Adenosine Deaminase Gene in Diploid Skin Fibroblasts from an Adenosine Deficient Human Proc Natl Acad Sci U S A 84 1055 1059 Perez P Tiraby G Kallerhoff J and Perret J 1989 Phleomycin Resistance as a Dominant Selectable Marker for Plant Cell Transformation Plant Mol Biol 13 365 373 Sambrook J Fritsch E F and Maniatis T 1989 Molecular Cloning A Laboratory Manual Second Ed Cold Spring Harbor Laboratory Press Plainview New York Sasaki Y Sone T Yahata K Kishine H Hotta J Chesnut J D Honda T and Imamoto F 2005 Multi gene gateway clone design for expression of multiple heterologous genes in living cells Eukaryotic clones containing two and three ORF multi gene cassettes expressed from a single promoter J Biotechnol 118 123 134 Sasaki Y Sone T Yoshida S Yahata K Hotta J Chesnut J D Honda T and Imamoto F 2004 Evidence for high specificity and efficiency of multiple recombination signals in mixed DNA cloning by the Multisite Gateway system J Biotechnol 107 233 243 Shigekawa
12. PBS pee ON E Resuspend cell pellet in a mixture of 20 ul of Resuspension Buffer and 2 ul of Lysis Solution CellsDirect Resuspension and Lysis Buffers see page xi Incubate the cell suspension at 75 C for 10 minutes o m Centrifuge for 1 minute to pellet cell debris Use 3 yl of the cell lysate to set up your PCR When performing PCR analysis on the genomic DNA isolated from your retargeted clones you should consider the following factors e We recommend using nested PCR with primary and secondary reactions to eliminate the high background observed with only primary PCR e Successful retargeting of your platform line genome introduces the human EF1a promoter upstream of the resistance gene to the selection marker resulting in blasticidin Geneticin or Zeocin resistance of successfully retargeted clones depending on the platform vector used during platform line creation You should design your primers from the EF1a promoter to the appropriate resistance marker You may use plasmid DNA or the Hygromycin resistance gene as a positive control e You may also design PCR primers specific to your gene s of interest in the retargeting construct to check for the presence of successful integrations e For a map and a description of the features of each platform vector pJTI Bsd pJTI Neo and pJTI Zeo and of the pJTI PhiC31 Int vector see pages 30 33 The vector sequences of pJTI Bsd pJTI Neo and pJTI Zeo ve
13. Zeocin e Vast increase in size similar to the effects of cytomegalovirus infecting permissive cells e Abnormal cell shape e Presence of large empty vesicles in the cytoplasm breakdown of the endoplasmic reticulum and Golgi apparatus or other scaffolding proteins e Breakdown of plasma and nuclear membrane appearance of many holes in these membranes Eventually these cells will completely break down and only strings of protein remain Zeocin resistant cells should continue to divide at regular intervals to form distinct colonies There should not be any distinct morphological changes in Zeocin resistant cells when compared to cells not under selection with Zeocin We have observed that stem cells display a considerably elevated sensitivity to selective antibiotics If you are retargeting platform cell lines generated from stem cells we recommend that you use a 10 fold lower range of concentrations for each of the selective agents when determining the sensitivity of your untransfected TM platform cell line to Blasticidin Geneticin and Zeocin Retargeting the R4 Platform Cell Line Introduction Method of Transfection Transfection Considerations The second step in targeted integration experiments is the retargeting event mediated by the R4 integrase expressed from pJTI R4 Int vector where the genetic elements of interest are site specifically integrated into the platform line genome when t
14. a second selection agent blasticidin neomycin or zeocin resistance genes in pJTI Bsd pJTI Neo or pJIT Zeo respectively but lacks the promoter to express from this resistance gene Transformants containing the desired R4 attP retargeting sequences and the promoterless selection marker are selected using Hygromycin B and expanded for the retargeting event Step 1 on the next page schematically depicts platform line creation The second step in targeted integration is the retargeting event mediated by the R4 integrase expressed from the pJTI R4 Int vector At this step the genetic elements of interest carried by the retargeting expression construct generated from pJTI R4 DEST using the MultiSite Gateway Pro Plus Vector Module see pages 14 18 are site specifically integrated into the platform line genome at the R4 attP target site introduced into the cell line at the first step This integration event also positions the constitutive human EF1a promoter upstream of the blasticidin neomycin or zeocin resistance gene i e promoterless selection marker thus allowing the selection of successfully retargeted transformants using the appropriate selection agent Step 2 on the next page depicts retargeting of the platform line For more information on PhiC31 and R4 Integrases and their uses in targeted integration refer to our website at www invitrogen com and published literature Thyagarajan et al 2008 Thyagarajan et
15. are well defined usually 5 days post microporation or 2 days post electroporation proceed to Selecting Retargeted Clones next page Continued on the next page 24 Retargeting the R4 Platform Cell Line continued Important Selecting Retargeted Clones Note To succefully select for your isogenic retargeted cell line you need to use the minimum concentration of the appropriate selective agent required to kill your untransfected mammalian R4 platform cell line See pages 19 22 for more information on detemining the sensitivity of your untransfected platform cell line to the selection agents After your cells have sufficiently recovered from transfection proceed with selection as described below Use the selection agent appropriate for the pJTI platform vector you have used to create your R4 platform cell line and incubate your cells in the suitable medium TM 1 48 to 72 hours after transfection or when the cells have sufficiently recovered and the colonies have become well defined transfer the cells into 100 mm dishes containing fresh medium Split cells such that they are no more than 25 confluent as the selection antibiotics work best at actively dividing cells 2 Incubate the cells at 37 C for 2 3 hours until they have attached sufficiently to the culture dish 3 Remove the medium and add fresh medium containing the appropriate selection agent at the proper concentration see Establishing Sensitivi
16. area in MEF medium with 2 5 ml per well of a gelatin coated 6 well dish 17 Freeze the cells for later use or use within 2 to 5 days after plating for hESC cell culture The medium should be changed every other day if they are not used immediately 41 Technical Support Web Resources Visit the Invitrogen website at www invitrogen com for e Technical resources including manuals vector maps and sequences application notes MSDSs FAQs formulations citations handbooks etc e Complete technical support contact information e Access to the Invitrogen Online Catalog e Additional product information and special offers Contact Us For more information or technical assistance call write fax or email Additional international offices are listed on our website www invitrogen com Corporate Headquarters Japanese Headquarters European Headquarters Invitrogen Corporation Invitrogen Japan Invitrogen Ltd 5791 Van Allen Way LOOP X Bldg 6F Inchinnan Business Park Carlsbad CA 92008 USA 3 9 15 Kaigan 3 Fountain Drive Tel 1 760 603 7200 Minato ku Tokyo 108 0022 Paisley PA4 9RF UK Tel Toll Free 1 800 955 6288 Tel 81 3 5730 6509 Tel 44 0 141 814 6100 Fax 1 760 602 6500 Fax 81 3 5730 6519 Tech Fax 44 0 141 814 6117 E mail tech_support invitrogen com E mail jpinfo invitrogen com MSDS Certificate of Analysis Limited Warranty 42 Material Safety Data Sheets MSDSs are available on our w
17. eR e n oh e P RR bates ions 26 Troubleshooting conet ie a eei ie mee qo RH e ht edible e in qns 27 Plu E n 30 PPE PSS IG adecuada MR oua a Za ele dfe oda Ea ott Cot mta tium mto 30 PITT Bd desee nd d e SM dE Nm du ben 31 PITE FEN CO arte Ae RR NINAKIEU NET A SUNT USO RT UTUNTUR NONO UNUEUENNS NUN DONSURUOUNUEDOP NOR I 32 PI TI VZ eO vei otn dmt MeL Y EN HE ET Lor t S Eo Ro 33 BITE BADEST A E b dabo dc a GM Masi oodd ended errr re trent ere terete 34 PILIS RUETIBE a osi ctt ea od o abdo Me m Elm Resolve TERY E DE 3b Assessing Gell Vitality eerie petere eite n ei hee te ba pde ee ar pen es 36 Freezing Mammalian Cells ser cte erre thee e un tested dete pee ien Pedes dena doen 37 Thawing Mammalian Cells ccccccccscsssssscsseseseseecenesesesesnssnesesesesueseseseseseseseseeeecesesesescananesesssesansnesesesesnenenees 39 Generating Mitomycin C Treated MEFS cccccscccssssesesssesnsnsesesescsneneseseeceesesesceceeseseseecenesesssesnananesesesnsnenenees 40 Technical Supporta kone a eE iiem tad e ict a ede bn SEI 42 Purchaser Notificatiort 5 erre eerte nter eerte entes een ces redet eei he tette pepe reed 43 Gateway Clone Distribution Poliey eoe D ce oles edd mde tuus t deus td grab SE Rh rig 45 Kefererces 5n en eedem de d te da mte dab ttd 46 iii iv Kit Contents and Storage Introduction System Components Shipping Storage Important This manual provides guidelines and in
18. pJTI R4 DEST as these strains are sensitive to CcdB effects For information on propagating and maintaining the pDONR vectors included in the MultiSite Gateway Pro Plus Vector Module refer top the MultiSite Gateway Pro manual supplied with Jump In TI Gateway System The MultiSite Gateway Pro manual is also available online at www invitrogen com or by contacting Technical Support see page 42 Preparation of Plasmid DNA For targeted integration experiments it is essential that the plasmid DNA used for transfection is of very high quality Typically best results have been obtained using plasmid DNA that has very low levels of endotoxins If using large quantities of DNA we recommend that the plasmid DNA is commercially prepared If smaller quantities are required use a commercial kit that delivers pure DNA that is free of endotoxins Follow the manufacturer s recommended protocol for DNA preparation Continued on next page General Information continued General Cell Handling Important When working with mammalian cells including stem cells handle as potentially biohazardous material under at least Biosafety Level 1 BL 1 containment For more information on BL 1 guidelines refer to Biosafety in Microbiological and Biomedical Laboratories 4 ed published by the Centers for Disease Control or see the following web site www cdc gov od ohs biosfty bmbl4 bmbl4toc htm For established cell lines e g
19. pJTI Bsd vector 6734 bp contains the PhiC31 attB site for PhiC31 integrase mediated integration into the genome of cell line of choice and the Hygromycin resistance gene under the control of Herpes simplex virus thymidine kinase promoter for subsequent selection It also contains the R4 attP site for R4 integrase mediated retargeting and the promoterless Blasticidin resistance gene for the selection of retargeted clones The vector sequence of pJTI Bsd is available from www invitrogen com or by contacting Technical Support see page 42 CET BIEC M T SV40 pA Features of pJTI Bsd 6734 nucleotides HSV TK bases 1 249 Hygromycin resistance gene 262 1296 HSV TK pA bases 1300 1790 pUC origin bases 1810 2483 c Ampicillin resistance gene bases 2631 3488 c PhiC31 attB integration site bases 3629 3907 c UMS terminator bases 4437 5467 R4 attP recombination site bases 5512 5575 Blasticidin resistance gene bases 5649 6047 SV40 pA bases 6205 6286 c complementary strand 31 pJTI Neo Map of pJTI Neo 32 The pJII Neo vector 7078 bp contains the PhiC31 attB site for PhiC31 integrase mediated integration into the genome of cell line of choice and the Hygromycin resistance gene under the control of Herpes simplex virus thymidine kinase promoter for subsequent selection It also contains the R4 attP site for R4 integrase mediated retargeting and the promoterless neomycin resistance gene for the sele
20. retargeting expression construct containing two DNA elements Refer to the MultiSite Gateway Pro manual 25 0942 supplied with the kit for detailed instructions PCR fragments BP reaction pDONR Vectors Entry Clones LR reaction Destination Vector Retargeting Expression att Clone Continued on next page 14 Constructing the Retargeting Expression Vector continued MultiSite Gateway Pro 3 Fragment Recombination Three PCR products flanked by specific attB or attBr sites and three MultiSite Gateway Pro Donor vectors are used in separate BP recombination reactions to TM generate three entry clones The three entry clones and the pJTI Fast DEST destination vector are used together in a MultiSite Gateway Pro LR recombination reaction to create your retargeting expression construct containing three DNA elements Refer to the MultiSite Gateway Pro manual 25 0942 supplied with the kit for detailed instructions LR reaction Destination Vector 2 3 Retargeting Expression atte attB2 Clone lt a Continued on next page 15 Constructing the Retargeting Expression Vector continued MultiSite Gateway Pro 4 Fragment Recombination MultiSite Gateway Pro Donor Vectors 16 Four PCR products flanked by specific attB or attBr sites and four MultiSite Gateway Pro Donor vectors are used in separate BP recombination reactions to generate two entry clones The four entry clones and the pJ
21. stand alone products Jump In TI Gateway Vector Kit Cat no A10896 and Jump In TI Platform Kit Cat no A10897 For more information on the Jump In TI Gateway System and its component kits visit our website at www invitrogen com or contact Technical Support page 42 Continued on next page Overview continued TM Jump In TI Gateway Targeted Integration Technology The Jump In TI Targeted Integration technology uses PhiC31 integrase mediated recombination to stably integrate DNA sequences of choice at specific genomic locations called pseudo attP sites in mammalian cells Unlike the better known recombinases such as Cre and Flp PhiC31 integrase catalyzes recombination between two non identical sites Further the lack of a corresponding excisionase enzyme makes the integration events catalyzed by PhiC31 unidirectional and virtually irreversible The Jump In TI Platform Kit as part of the Jump In TI Gateway System places a target in the chromosomal DNA for a second site specific integration event mediated by the R4 Integrase i e retargeting The first step in targeted integration is the creation of the R4 platform line This is accomplished by the PhiC31 integrase mediated site specific insertion of the R4 integrase target sequences i e attP along with the Hygromycin resistance gene from a pJTI platform vector The pJTI platform vector also contains the sequences for resistance against
22. the buyer is an academic or for profit entity The buyer cannot sell or otherwise transfer a this product b its components or c materials made using this product or its components to a third party or otherwise use this product or its components or materials made using this product or its components for Commercial Purposes The buyer may transfer information or materials made through the use of this product to a scientific collaborator provided that such transfer is not for any Commercial Purpose and that such collaborator agrees in writing a not to transfer such materials to any third party and b to use such transferred materials and or information solely for research and not for Commercial Purposes Commercial Purposes means any activity by a party for consideration and may include but is not limited to 1 use of the product or its components in manufacturing 2 use of the product or its components to provide a service information or data 3 use of the product or its components for therapeutic diagnostic or prophylactic purposes or 4 resale of the product or its components whether or not such product or its components are resold for use in research For products that are subject to multiple limited use label licenses the terms of the most restrictive limited use label license shall control Life Technologies Corporation will not assert a claim against the buyer of infringement of patents owned or controlled by Life Technologies
23. thus making the integration event unidirectional and ensuring that the constructs integrated into the genome do not act subtsrates for the reverse reaction Therefore the Hygromycin B resistance conferred to your cell line by the integration of the platform vector and the subsequent selection in selective medium virtually guarantees that your clones contain the R4 retargeting sequence However you may still screen your expanded clones by Southern blot analysis to ascertain that only a single integration event has taken place and by PCR analysis for the presence of R4 retargeting sequences You can use Southern blot analysis to determine the number of integrations in each of your Hygromycin B resistant clones When performing Southern blot analysis you should consider the following factors e Probe We recommend that you use a fragment of the Hygromycin resistance gene 1 kb as the probe to screen your samples You may amplify the Hygromycin expression cassette from one of the pJTI platform vectors using the appropriate primers To label the probe we generally use a standard random priming kit e g Ambion DECAprime IT Kit Cat no 1455 Other random priming kits are suitable e Genomic DNA We recommend using the DNAzol Reagent see page xi to isolate the genomic DNA from the Hygromycin B resistant clones e Restriction digest When choosing a restriction enzyme to digest the genomic DNA we recommend choosing an enzyme that
24. xii for ordering information e Hygromycin B is light sensitive Store the liquid stock solution at 4 C protected from exposure to light e Hygromycin B is toxic Do not ingest solutions containing the drug e Wear gloves a laboratory coat and safety glasses or goggles when handling Hygromycin B and Hygromycin B containing solutions Follow the instructions provided with Hygromycin B to prepare your working stock solution The stability of Hygromycin B is guaranteed for six months if stored at 4 C in the dark Medium containing Hygromycin B is stable for up to six weeks Continued on next page Generating the R4 Platform Cell Line continued Determining the To successfully generate an R4 platform cell line containing the R4 attP Hygromycin B retargeting sequence you need to determine the minimum concentration of Sensitivity Hygromycin B required to kill your untransfected cells Typically concentrations ranging from 10 to 400 ug ml of Hygromycin B are sufficient to kill most untransfected mammalian cell lines We recommend that you test a range of concentrations see protocol below to determine the minimum concentration necessary for your cell line of choice 1 Plate or split a confluent plate so that the cells will be approximately 25 confluent Prepare a set of 7 plates Allow cells to adhere overnight 2 The next day substitute culture medium with medium containing varying concentrations of Hygromycin B 0
25. 08 Phone 760 603 7200 Fax 760 602 6500 email outlicensing invitrogen com EF lalpha promoter products are sold under license for research purposes only The use of this product for any commercial purpose including but not limited to use in any study for the purpose of a filing of a new drug application requires a license from Mochida Pharmaceutical Co Ltd 7 Yotsuya 1 Chome Shinjuku Ku Tokyo 160 Japan Tel 81 3 3225 5451 Fax 81 3 3225 6091 This product and its use are the subject of one or more of U S Patent Nos 6 632 672 and 7 361 641 and foreign equivalents This product or one or more vectors made using this product is the subject of U S Patent No 5 888 732 owned by Life Technologies Corporation Gateway Clone Distribution Policy Introduction Gateway Entry Clones Gateway Expression Clones Additional Terms and Conditions The information supplied in this section is intended to provide clarity concerning Invitrogen s policy for the use and distribution of cloned nucleic acid fragments including open reading frames created using Invitrogen s commercially available Gateway Technology Invitrogen understands that Gateway entry clones containing attL1 and attL2 sites may be generated by academic and government researchers for the purpose of scientific research Invitrogen agrees that such clones may be distributed for scientific research by non profit organizations and by for prof
26. 1 P1P5r and pDONR221 P5P2 3 pDONR221 P1P4 pDONR221 P4rP3r and pDONR221 P3P2 4 pDONR221 P1P5r pDONR221 P5P4 pDONR221 P4rP3r and pDONR221 P3P2 Continued on next page Constructing the Retargeting Expression Vector continued pJTI R4 DEST Destination Vector Recombination Region of pJTI R4 DEST TM The pJTI R4 DEST vector is specifically designed to be used in a MultiSite Gateway Pro LR recombination reaction to create your retargeting expression clone to site specifically integrate your multiple DNA elements into the genome of your R4 platform cell line The pJIT R4 DEST vector contains the constitutive human EF1a promoter which when integrated upstream of the promoterless resistance gene by the R4 Integrase results in Blasticin Geneticin or Zeocin resistance of the successfully retargeted clones For a map and features of pJII R4 DEST see page 34 The recombination region of the retargeting expression clone resulting from pJTI R4 DEST x pDONR entry clone is shown below Shaded regions correspond to those DNA sequences recombinationally transferred from the entry clone into pJTI R4 DEST vector Non shaded regions are derived from the pJTI R4 DEST vector The vector sequence of pJIT R4 DEST is available on our website at www invitrogen com or by contacting Technical Support see page 42 3663 TATGTTGTGT GGAATTGTGA GCGGATAACA ATTTCACACA GGAAACAGCT ATGACCATGA TTACGCCAAG CTTGCATGCC TGCAGGTCGA CTCT
27. 10 50 100 200 400 600 ug ml 3 Replenish the selective media every 3 4 days and observe the percentage of surviving cells 4 Note the percentage of surviving cells at regular intervals to determine the appropriate concentration of Hygromycin B that kills the cells within 1 2 weeks after the addition of Hygromycin B Method of For established cell lines consult original references or the supplier of your cell Transfection line for optimal method of transfection Methods of transfection include lipid mediated transfection Felgner et al 1989 Felgner amp Ringold 1989 calcium phosphate precipitation Chen amp Okayama 1987 Wigler et al 1977 and electroporation Chu et al 1987 Shigekawa amp Dower 1988 We have achieved satisfactory results with two nonviral gene delivery methods lipid mediated transfection using Lipofectamine 2000 see page xi for ordering information and electroporation or microporation Both methods do not seem to affect the growth characterisitics of the cells however certain variant stem cell lines are refractory to transfection by Lipofectamine 2000 Note that if you use calcium phosphate or lipid mediated transfection methods the amount of total DNA required for transfection is typically higher than for electroporation We have obtained the best results using high efficiency transfection methods such as microporation or electroporation and we recommend that you use these methods as
28. 61 200 mM L Glutamine 100 ml 25030 081 MEM Non Essential Amino Acids Solution 10 mM 100 ml 11140 050 100X HT Supplement 50 ml 11067 030 bFGF FGF Basic Human Recombinant 50ug PHG0026 Fetal Bovine Serum Certified 500 ml 16000 044 Fetal Bovine Serum Qualified 500 ml 26140 079 Fetal Bovine Serum ES Cell Qualified US 500 ml 16141 079 Pluronic F 68 10 100X 100 ml 24040 032 Knockout Serum Replacement KSR 500 ml 10828 028 Bovine Albumin Fraction V Solution 7 596 100 ml 15260 037 BSA 10 Ultrapure Molecular Biology Grade 1000 ml P2458 2 Mercaptoethanol 50 ml 21985 023 Invitrogen also provides ES Cell Qualified Fetal Bovine Serum originating from countries other than the US These can be more appropriate for your situation and may be used to maintain your stem cell culture For more information refer to www invitrogen com Mitomycin C treated Hygromycin resistant primary MEFs are available from Millipore Cat no PMEF H or ATCC SCRC 1045 2 Hygromycin resistant primary MEF that are not Mitomycin treated are also available separately from Millipore Cat no PMEF HL or ATCC Cat no SCRC 1045 One vial of cells 5 x 106 6 x 10 cells vial can be used to plate ten 60 mm dishes MEFs which are not mitotically arrested must be treated with Mitomycin C before use Mitomycin C is available separately from Sigma St Louis Cat no M4287 Porcine Skin Gelatin can be obtained from Sigma St Louis
29. AGATCT ATACAACACA CCTTAACACT CGCCTATTGT TAAAGTGTGT CCTTTGTCGA TACTGGTACT AATGCGGTTC GAACGTACGG ACGTCCAGCT GAGATCTAGA 3785 5466 3763 GCAGAATTCG GCTTACCACT TTGTACAAGA AAGCTGGGTN GENE S NNAGOCTGCTOTTTTTGTACA AACTTGTAAG CCGAATTCCA GCACACTGGC CGTCTTAAGC CGAATGGTGA AACATGTTCE TECGACCCTN 2 4 NNTCGGACGA AAAAACATGT TTGAACATTC GGCTTAAGGT CGTGTGACCG l f l l attB2 attB1 Important Preparing Plasmid DNA For targeted integration experiments it is essential that the plasmid DNA used for transfection is of very high quality Typically best results have been obtained using plasmid DNA that has very low levels of endotoxins If using large quantities of DNA we recommend that the plasmid DNA is commercially prepared If smaller quantities are required use a commercial kit that delivers pure DNA that is free of endotoxins Follow the manufacturer s recommended protocol for DNA preparation Continued on next page 17 Constructing the Retargeting Expression Vector continued Generating Entry Clones Generating Retargeting Expression Clones 18 Ensure that primers used for PCR amplification are of good quality Since these primers are generally 45 bases in length the possibility of mutations is greater Mutations in the PCR primers may in turn lead to inefficient recombination with the pDONR vectors If possible avoid using a plasmid containing the kanamycin resistance gene as the template for PCR If the f
30. Cat no G1890 Continued on next page Accessory Products continued Additional Products For more information on the following accessory products refer to our website at www invitrogen com or contact Technical Support see page 42 Product Amount Cat no Trypsin EDTA 0 05 Trypsin EDTAe4Na 100 ml 25300 054 1X liquid 20x 100 ml 25300 120 Versene EDTA 0 05 Versene EDTAe4Na 100 ml 15040 066 1X liquid TrypLE Express Dissociation Enzyme without 100 ml 12604 013 Phenol Red 20 x 100 ml 12604 039 Antibiotic Antimycotic 100X liquid 100 ml 15240 062 Penicillin Streptomycin 100 ml 15070 063 Lipofectamine 2000 Transfection Reagent 1 5 ml 11668 019 15 ml 11668 500 Geltrex 5 ml 12760 021 Geltrex hESC qualified 1ml A10480 01 Collagenase Type IV 1g 17104 019 StemPro EZChek Human Tri Lineage 100 reactions 23191 050 Multiplex PCR Kit StemPro EZPassage Disposable Stem Cell 10 tools 23181 010 Passaging Tool disposable Anti Clumping Agent 20 ml 01 0057AE LIVE DEAD Cell Vitality Assay Kit 1000 assays L34951 Trypan Blue Stain 100 ml 10250 061 ProLong Gold Antifade Reagent 10 ml P36930 ProLong Gold Antifade Reagent with DAPI 10 ml P36931 CellsDirect Resuspension and Lysis Buffers 1 kit 11739 010 AccuPrime Taq DNA Polymerase High Fidelity 1000 12346 094 reactions DNAzol Reagent 100 ml 10503 027 HEPES Buffer Solution
31. Corporation which cover this product based upon the manufacture use or sale of a therapeutic clinical diagnostic vaccine or prophylactic product developed in research by the buyer in which this product or its components was employed provided that neither this product nor any of its components was used in the manufacture of such product If the purchaser is not willing to accept the limitations of this limited use statement Life Technologies is willing to accept return of the product with a full refund For information about purchasing a license to use this product or the technology embedded in it for any use other than for research use please contact Out Licensing Life Technologies 5791 Van Allen Way Carlsbad California 92008 or outlicensing lifetech com Continued on next page 43 Purchaser Notification continued Limited Use Label License No 51 Blasticidin and the Blasticidin Selection Marker Limited Use Label License No 60 EF1alpha Promoter Limited Use Label License No 328 Phi C31 Recombinase Technology Limited Use Label License No 345 Gateway Vec tors 44 Blasticidin and the blasticidin resistance gene bsd are the subject of U S Patent No 5 527 701 sold under patent license for research purposes only For information on purchasing a license to this product for purposes other than research contact Licensing Department Life Technologies Corporation 5791 Van Allen Way Carlsbad California 920
32. HeLa COS 1 consult original references or the supplier of your cell line for detailed instructions on maintaining your cells and the optimal method of transfection Pay particular attention to the exact medium requirements when to passage the cells and at what dilution to split the cells The guidelines below are general instructions that pertain to many cell lines for best results we recommend that you follow the protocols of your cell line exactly e All solutions and equipment that come in contact with the cells must be sterile Always use proper aseptic technique and work in a laminar flow hood Before starting experiments be sure to have your cells established at least 5 passages and also have at least 10 20 vials of frozen stocks on hand We recommend using early passage cells for your experiments e For general maintenance of cell culture passage your cells when they are near confluence gt 80 90 confluent Avoid overgrowing cells before passaging e Use Trypan Blue exclusion or the LIVE DEAD Cell Vitality Assay Cat no L34951 to determine cell viability Log phase cultures should be gt 90 viable When thawing or subculturing transfer your cells into pre warmed medium e 10 pl ml of antibiotic antimycotic containing penicillin streptomycin and amphotericin B may be used if required see page xi for ordering information e Cells should be at the appropriate confluence usually 70 90 confluency in a 60 mm d
33. Int vector 5705 bp contains the gene for R4 Integrase from the Steptomyces PhiC31 phage The R4 Integrase allows the site specific integration of DNA elements into the genome of the platform cell line from the pJTI R4 DEST retargeting expression construct upon cotransfection of the platform line with both vectors The vector sequence of pJTI R4 Int is available from www invitrogen com or by contacting Technical Support see page 42 pJTI R4 Int 5705 bp S 2 Q lt Features of pJTI R4 Int 5705 nucleotides T7 promoter bases 1 20 HCO R4Int bases 43 1452 c CMV promoter bases 1590 2113 c pUC origin bases 2598 3271 Ampicillin resistance gene ORF bases 3458 4318 c SV40 polyA site bases 5254 5616 c c complementary strand 35 Assessing Cell Vitality Introduction LIVE DEAD Cell Vitality Assay 36 We recommend using the LIVE DEAD Cell Vitality Assay Kit available separately from Invitrogen to assess the vitality of your cells by flow cytometry For more information on how to distinguishes metabolically active cells from cells that are dead or injured refer to the manual provided with the LIVE DEAD Cell Vitality Assay Kit Cat no L34951 For ordering information see page xi The assay has been optimized using Jurkat cells Some modifications may be required for use with other cell types A negative control for necrosis should be prepared by incubating cells wi
34. Invitrogen Jump In TI Gateway Targeted Integration System MultiSite Gateway adapted Vector System for Generation of Isogenic Stable Mammalian Cell Lines Catalog nos A10895 A10896 and A10897 Version C 7 June 2010 A10900 ii Table of Contents Table of Contents iod cheat tense tee diem Sardi ETE IE de P les tris bites ener eon ere ees EE iii Kit Contents and Storage csset dete seio ote de Mien ep eire eI gea v Accessory Products o ee E EA AE NEE E AE e i ee e dee eite ix Introduction ase sess tests hcctu creates cates a ctesatecetase snes sOedaesdeacecoteter iebleccedsiinc nnne anenun annann annann 1 OVERVIEW oes dducstusce bodies tetendit doti seii ente eet ete ed dub e bundles ot et toasters 1 MethodsS ge M M 5 General Information o meo ete detiene e epit o aed Whe AH ad dle fea ee hn SO te doe caer 5 Generating the R4 Platform Cell Linero E EE RE nennen tenente tnnt 7 Screening R4 Platform Cell Line Clones iseis re iii aea E A EE E E R ttn 11 Determining Site of Integration sssssssssssssssseseeeenenene tenente nennt 13 Constructing the Retargeting Expression Vector ccccesssesescsssssescsceceseseeeeceesesesesnsnenesesesesnensisseseeeesenesees 14 Establishing Sensitivity to Selection Agents sse 19 Retargeting the R4 Platform Cell Line sisisi iieii eei eriet teisi tereni erneiere ittee iiris iet se 23 Screening Retargeted Clones ie er RUE e P ie ine n
35. TI Fast DEST destination vector are used together in a MultiSite Gateway Pro LR recombination reaction to create your retargeting expression construct containing four DNA elements Refer to the MultiSite Gateway Pro manual 25 0942 supplied with the kit for detailed instructions PCR fragments BP reaction pDONR Vectors Y Y v V Entry Clones en ee a V a LR reaction attL1 A Retargeting Expression attB1 en attBS B4 E attB2 lone Destination Vector The MultiSite Gateway Pro donor vectors are used to clone attB or attBr flanked PCR products to generate entry clones and contain similar elements as other Gateway donor vectors However because different attB sites will flank your PCR products different donor vectors are required to facilitate generation of entry clones which are later used in creating your retargeting expression construct The table below lists the specific donor vectors required to assemble a retargeting expression construct containing one two three or four DNA elements of interest For a map and a description of the features of each MultiSite Gateway Pro donor vector refer to the MultiSite Gateway Pro manual 25 0942 supplied with the kit Note pDONR 221 is provided as a positive control for the BP recombination reaction and should not be used to generate multi fragment entry clones bcd ar Donor Vectors Required ragments 1 pDONR201 or pDONR221 2 pDONR22
36. al 2001 In addition to the Jump In TI Gateway System which enables the rapid creation of isogenic stable cell lines Invitrogen also offers the Jump In Fast Gateway System Cat no A10893 which facilitates the generation of a polyclonal pool of mammalian cells that over expresses your protein of interest In the Jump In Fast Gateway System your gene of interest is directly inserted into the genome in a single recombination step mediated by the PhiC31 integrase For more information on Jump In TI Gateway and Jump In Fast Gateway Systems visit our website at www invitrogen com or contact Technical Support page 42 Continued on next page Overview continued Jump In TI The schematic below depicts the major steps of the targeted integration reaction Gateway System using theJump In TI Gateway System Workflow promoterless Bsd NeoR or ZeoR PhiC31 Int R4 attP pTK pJTI Hyg PhiC31 attB PhiC31 pseudo attP TVVP ONN Mammalian Genome l pTK Hyg promoterless R4 attP BsdR NeoR or ZeoR Platform Cell Line Genome Step 1 Platform Creation Gene s of Interest R4 Int Expression Construct R4 attB 4 pTK HygR promoterless R4 attP BsdR NeoR or ZeoR Platform Cell Line Genome BsdR NeoR EF1a Gene s of or ZeoR Interest Retargeted Genome Step 2 Retargeting Continued on next page Overview continued Purpose of This Manual Important
37. ate ten 60 mm dishes MEFs which are not mitotically arrested must be treated with Mitomycin C before use Continued on next page Generating Mitomycin C Treated MEFs continued Mitomycin C Inactivation Use the procedure below to generate mitotically inactivated MEFs in T175 culture flasks Make sure that the MEFs to be treated with Mitomycin C are 90 95 confluent in T175 flasks 3 days after the initial thawing Observe each flask individually under the microscope to ensure cell growth and culture sterility 1 Culture MEFs in MEF medium see page 40 for recipe 2 Ina biosafety cabinet aspirate the medium from T175 flasks and add 16 ml of Mitomycin C solution 10 pg ml 3 Incubate MEFs treated with 10 ug ml Mitomycin C in the flasks for 2 3 hours at 37 C 5 CO Work in sets of no more than six flasks at a time 4 After 2 3 hours of incubation aspirate off the Mitomycin C solution and neutralize the waste with bleach see above 5 Wash cells five times with Dulbecco s Phosphate Buffered Saline D PBS containing Mg and Ca see page ix for ordering information 6 Aspirate D PBS and wash cells with 20 ml D PBS that is Mg and Ca free see page ix ordering information 7 Add3 ml of 0 05 Trypsin EDTA solution per flask to trypsinize cells see page xi for ordering information At room temperature monitor the degree of cell detachment while gently rocking and tapping the flask Note MEFs are trypsin
38. ations or documentation If you discover an error in any of our publications please report it to our Technical Support Representatives Invitrogen assumes no responsibility or liability for any special incidental indirect or consequential loss or damage whatsoever The above limited warranty is sole and exclusive No other warranty is made whether expressed or implied including any warranty of merchantability or fitness for a particular purpose E mail eurotech invitrogen com Purchaser Notification Introduction Information for European Customers Limited Use Label License No 5 Invitrogen Technology Use of the StemPro TARGET hESC BGOIv Kit is covered under the licenses detailed below StemPro TARGET hESC BG01v cells variant hESC BGOIV are genetically modified and carry a chromosomal target site for R4 Integrase and a Hygromycin Resistance gene The paternal human stem cells were derived March 2001 from a supernumerary IVF embryo that would have otherwise been discarded and was obtained with informed consent As a condition of sale this product must be in accordance with all applicable local legislation and guidelines including EC Directive 90 219 EEC on the contained use of genetically modified organisms The purchase of this product conveys to the buyer the non transferable right to use the purchased amount of the product and components of the product in research conducted by the buyer whether
39. c needs and requirements of your particular cell line as these vary considerably between different cell lines and under different laboratory conditions This manual does not provide instructions for generating the retargeting construct using MultiSite Gateway Technology For instructions on designing and creating the retargeting construct refer to the MultiSite Gateway Pro manual 25 0942 supplied with the kit For more information on the MultiSite Gateway Technology and general cell culture maintenance visit our website at www invitrogen com or contact Technical Support see page 42 TM To propagate and maintain the pJIT R4 DEST vector we recommend using 10 ng of the vector to transform One Shot ccdB Survival 2 T1 Chemically Competent Cells see page xii from Invitrogen The ccdB Survival 2 T1 E coli strain is resistant to CcdB effects and can support the propagation of plasmids containing the ccdB gene To propagate and maintain the pJIT R4 Int pJTI PhiC31 Int pJTI Bsd pJTI Neo and pJTI Zeo vectors we recommend using 10 ng of each vector to separately transform a recA endA E coli strain like TOP10F DH5a T1 TOP10 or equivalent Select transformants on LB plates containing 50 100 ug ml ampicillin Be sure to prepare a glycerol stock of a transformant containing plasmid for long term storage Note Do not use general E coli cloning strains including TOP10 or DH5a for propagation and maintenance of
40. ction of retargeted clones with Geneticin The vector sequence of pJ1T Neo is available from www invitrogen com or by contacting Technical Support see page 42 R4 attP Neomycin SV40 pA Features of pJTI Neo 7078 nucleotides HSV TK bases 1 249 Hygromycin resistance gene 262 1296 HSV TK pA bases 1300 1790 pUC origin bases 1810 2483 c Ampicillin resistance gene bases 2631 3488 c PhiC31 attB integration site bases 3629 3907 c UMS terminator bases 4437 5467 R4 attP recombination site bases 5512 5575 Neomycin resistance gene bases 5582 6376 SV40 pA bases 6550 6680 c complementary strand pJTI Zeo Map of pJTI Zeo The pJ II Zeo vector 6597 bp contains the PhiC31 attB site for PhiC31 integrase mediated integration into the genome of cell line of choice generating the platform cell line and the Hygromycin resistance gene under the control of Herpes simplex virus thymidine kinase promoter for selection It also contains the R4 attP site for R4 integrase mediated retargeting and the promoterless Zeocin resistance gene for the selection of retargeted clones The vector sequence of pJTI Zeo is available from www invitrogen com or by contacting Technical Support see page 42 R4 attP Zeocin SV40 pA Features of pJTI Zeo 6597 nucleotides HSV TK bases 1 249 Hygromycin resistance gene 262 1296 HSV TK pA bases 1300 1790 pUC origin bases 1810 2483 c Ampicillin resistance
41. ctors are available on our website at www invitrogen com or by contacting Technical Support see page 42 e We recommend a high fidelity thermostable DNA polymerase such as the AccuPrime Tag DNA Polymerase see page xi for the nested PCR e Be sure to include a final extension step 7 minutes at 72 C in your PCR PCR is usually sufficient to confirm the presence of the retargeted sequences in your cell line after transfection However you may also perform a Southern blot analysis as an additional check to screen for a single copy number Use the Southern blot protocol of your choice with a radiolabeled probe from the expression vector used to retarget the cells We recommend using the DNAzol Reagent see page xi to isolate genomic DNA from the platform cell line Troubleshooting Introduction Culturing Cells The following tables list some potential problems and possible solutions to help you troubleshoot your targeted integration experiments For troubleshooting any potential problems that might arise when generating your retargeting expression construct refer to the MultiSite Gateway Pro manual 25 0942 supplied with the kit The table below lists some potential problems and solutions that help you troubleshoot your cell culture problems Problem Cause Solution No viable cells after thawing stock Stock not stored correctly Order new stock and store in liquid nitrogen Keep in liquid nitrog
42. cuts at a single known site outside of the Hygromycin resistance gene in the pJTI platform vector used such as BamH I or Hind III Hybridization of the Hygromycin probe to the digested DNA should then allow you to detect a single band containing the Hygromycin resistance gene from pJTI platform vector if only one integration event has occurred e Protocol You may use any Southern blotting protocol of your choice Refer to Current Protocols in Molecular Biology Ausubel et al 1994 or Molecular Cloning A Laboratory Manual Sambrook et al 1989 for detailed protocols If you digest genomic DNA from your transfectants with an appropriate restriction enzyme that cuts at a single known site outsite the Hygromycin resistance gene and use a Hygromycin resistance gene fragment as a probe in your Southern analysis you should be able to easily distinguish between single and multiple integration events DNA from single integrants should contain only one hybridizing band TM corresponding to a single copy of the integrated pJII platform vector DNA from multiple integrants should contain more than one hybridizing band If the pJTI platform vector integrates into multiple chromosomal locations the bands may be of varying sizes Continued on next page 11 Screening R4 Platform Cell Line Clones continued PCR Analysis Preparation of Genomic DNA for PCR What You Should See Freezing R4 Platform Cells 12
43. determine the minimum concentration of the selective agent required to kill your untransfected mammalian R4 platform cells This section provides instructions for establishing the sensitivity of your platform cell line to each of the selection agents TM The pJIT Bsd platform vector contains the Blasticidin S deaminase gene for the selection of transfectants with the antibiotic Blasticidin The deaminase converts Blasticidin S to a nontoxic deaminohydroxy derivative Izumi et al 1991 Blasticidin S HCl is available separately from Invitrogen see page xii e Blasticidin S is toxic Do not ingest solutions containing the drug e Wear gloves a laboratory coat and safety glasses or goggles when handling Blasticidin S and Blasticidin S containing solutions e Always weigh Blasticidin S and prepare solutions in a hood Follow the instructions provided with Blasticidin S to prepare your working stock solution Aliquot in small volumes suitable for one time use and store at 4 C short term or at 20 C long term Do not subject stock solutions to freeze thaw cycles and do not store in a frost free freezer Aqueous stock solutions are stable for 1 2 weeks at 4 C and 6 8 weeks at 20 C Medium containing Blasticidin may be stored at 4 C for up to 2 weeks The Blasticidin concentration required for selection in mammalian cells varies depending on the cell line used Use 2 10 ug ml Blasticidin for selection in mammalian cell
44. ebsite at www invitrogen com msds The Certificate of Analysis CofA provides detailed quality control information for each product The CofA is available at www invitrogen com support and is searchable by product lot number which is printed on each box Invitrogen is committed to providing our customers with high quality goods and services Our goal is to ensure that every customer is 10076 satisfied with our products and our service If you should have any questions or concerns about an Invitrogen product or service contact our Technical Support Representatives Invitrogen warrants that all of its products will perform according to specifications stated on the certificate of analysis The company will replace free of charge any product that does not meet those specifications This warranty limits Invitrogen Corporation s liability only to the cost of the product No warranty is granted for products beyond their listed expiration date No warranty is applicable unless all product components are stored in accordance with instructions Invitrogen reserves the right to select the method s used to analyze a product unless Invitrogen agrees to a specified method in writing prior to acceptance of the order Invitrogen makes every effort to ensure the accuracy of its publications but realizes that the occasional typographical or other error is inevitable Therefore Invitrogen makes no warranty of any kind regarding the contents of any public
45. en until thawing Home made stock not viable Freeze cells at a density of 2 x 109 3 x 10 viable cells ml Use low passage cells to make your own stocks Follow the freezing procedure for your type of cell culture exactly Slow freezing and fast thawing are crucial Add the cold freezing medium in a dropwise manner slowly swirling the tube after each drop At the time of thawing thaw quickly and do not expose vial to the air but quickly change from nitrogen tank to 37 C water bath Obtain new cells Thawing medium not correct Use specified medium Cells too diluted Generally we recommend thawing one vial in a 35 mm dish If you need to concentrate cells spin down the culture for 4 minutes at 200 x g at room temperature and dilute the cells at higher density MEFs sub optimal and do not support recovery of your stem cells if using stem cells thawed on MEF feeders Purchase or make a new batch of mitotically inactivated MEFs see page 36 MEFs overgrow plate MEFs not inactivated Inactivate mitosis in MEFs as described on pages 34 41 or purchase inactivated MEFs see page x Continued on next page 27 Troubleshooting continued Culturing Cells The table below lists some potential problems and solutions that help you troubleshoot your cell culture problems Problem Cause Solution Cells grow slowly Growth medium not correct
46. ering vectors in the simplest most direct fashion as well as to perform transfection and selection procedures to generate your recombinant cell line expressing your gene s of interest in the most efficient way the use of these products is geared towards users who are familiar with the concepts of the Gateway Technology site specific recombination and culturing mammalian and stem cells If you are unfamiliar with these technologies we recommend that you acquire a working knowledge of the Gateway Technology and mammalian and stem cell culture Continued on next page V Kit Contents and Storage continued Kit Components Jump In TI Platform Kit Jump In TI Gateway Vector Kit vi TM TM The Jump In TI Gateway System contains the following components The contents of each kit component are described below The Jump In TI Platform Kit supplied with the Jump In TI Gateway System is also available individually Cat no A10897 It contains the vectors used for platform cell line generation Store the vectors at 20 C Vector Composition Amount pJIT Bsd 20 ul of vector at 500 ng pl in TE buffer pH 8 0 10 ug pJIT Neo 20 ul of vector at 500 ng pl in TE buffer pH 8 0 10 ug pJII Zeo 20 ul of vector at 500 ng pl in TE buffer pH 8 0 10 pg pJTI PhiC31 Int 20 pl of vector at 500 ng pl in TE buffer pH 8 0 10 pg TE buffer pH 8 0 10 mM Tris HCl 1 mM EDTA pH 8 0
47. es Do not use lower concentrations of collagenase and treat for longer periods Cells passaged too early Passaging stem cells too early causes poor plating and differentiation Grow to cells to near confluence i e a day or two longer than when the colonies are just touching No growth after transfection Incorrect amount of selection agent is used Determine the minimum concentration of the selection agent required to kill untransfected cells as described on page 8 and pages 19 22 and use this amount for selection 28 Continued on next page Troubleshooting continued Transfecting Cells The table below lists some potential problems and solutions that help you troubleshoot your problems during transfection Problem Cause Solution Low survival rate after transfection Poor DNA quality The quality of the plasmid DNA strongly influences the results of transfection experiments Use endotoxin free DNA for all transfections Make sure that the A260 A280 ratio of the DNA is between 1 8 and 2 0 Do not use phenol chloroform extraction or ethanol precipitation Cells are cultured in suboptimal conditions Cells that are 80 90 confluent are ideal for transfection A higher confluency often results in a higher proportion of dead cells in culture Avoid excessive cell densities of high confluency Cells are harvested from selective plates prior to transfection for reta
48. f Zeocin required to kill your untransfected R4 platform cell line Typically concentrations ranging from 50 to 1000 ug ml Zeocin are sufficient to kill most untransfected mammalian cell lines with the average being 100 to 400 ug ml We recommend that you test a range of concentrations to ensure that you determine the minimum concentration necessary for your cell line 1 Plate or split a confluent plate so that the cells will be approximately 25 confluent Prepare a set of 7 plates Allow cells to adhere overnight 2 The next day substitute culture medium with medium containing varying concentrations of Zeocin 0 50 100 250 500 750 and 1000 ug ml Zeocin 3 Replenish the selective media every 3 4 days and observe the percentage of surviving cells 4 Note the percentage of surviving cells at regular intervals to determine the appropriate concentration of Zeocin that kills the cells within 1 2 weeks TM after addition of Zeocin Continued on next page 21 Establishing Sensitivity to Selection Agents continued Effect of Zeocin on Sensitive and Resistant Cells Important 22 TMI Zeocin s method of killing is quite different from other antibiotics including Hygromycin B Geneticin G 418 and blasticidin Cells exposed to fatal concentrations of Zeocin do not round up and detach from the plate Sensitive TM cells may exhibit the following morphological changes upon exposure to
49. form Kit for the generation of a stable platform cell line that can later be retargeted using an expression construct containing your genetic elements of interest The Jump In TI Platform Kit consists of three platform vectors pJTI Bsd pJTI Neo and pJTI Zeo expressing the blasticidin neomycin and zeocin resistance markers respectively and the pJIT PhiC31 Int vector that expresses the PhiC31 Integrase For a map and features of each vector see pages 30 33 e The MultiSite Gateway Pro Plus Vector Module for simultaneous cloning of up to four DNA fragments to generate a retargeting construct Based on the Gateway Technology Hartley et al 2000 Sasaki et al 2005 Sasaki et al 2004 the MultiSite Gateway uses site specific recombinational cloning to allow simultaneous cloning of multiple DNA fragments in a defined order and orientation e The Jump In TI Gateway Vector Kit for retargeting of platform cell lines The Jump In TI Gateway Vector Kit consists of the pJTI R4 DEST vector i e the retargeting construct when containing your DNA elements of interest and the pJTI R4 Int vector expressing the R4 Integrase For a map and features of each vector see pages 34 35 For the recombination region of the pJTI R4 DEST see page 17 In addition to the complete Jump In TI Gateway System Cat no A10895 containing all the components listed above Invitrogen also offers the individual component kits as
50. ge activity Expression of this protein in eukaryotic and prokaryotic hosts confers resistance to Zeocin Calmels et al 1991 Drocourt et al 1990 Zeocin is a member of the bleomycin phleomycin family of antibiotics isolated from Streptomyces Antibiotics in this family are broad spectrum antibiotics that act as strong anti bacterial and anti tumor drugs They show strong toxicity against bacteria fungi including yeast plants and mammalian cells Baron et al 1992 Drocourt et al 1990 Mulsant et al 1988 Perez et al 1989 Zeocin is available separately from Invitrogen see page xii for ordering information e Zeocin is light sensitive Store Zeocin plates and medium containing Zeocin in the dark e Wear gloves a laboratory coat and safety glasses or goggles when handling solutions containing Zeocin e Zeocin is toxic Do not ingest or inhale solutions containing the drug TM Follow the instructions provided with Zeocin to prepare your working stock solution For your convenience the drug is prepared in autoclaved deionized water and is available in 1 25 ml aliquots at a concentration of 100 mg ml Store Zeocin at 20 C in the dark and thaw on ice before use The stability of Zeocin is guaranteed for six months if stored properly To successfully retarget your platform cell line containing the promoterless zeocin resistance gene you need to determine the minimum concentration o
51. gene bases 2631 3488 c PhiC31 attB integration site bases 3629 3907 c UMS terminator bases 4437 5467 R4 attP recombination site bases 5512 5575 Zeocin resistance gene bases 5659 6030 SV40 pA bases 6047 6128 c complementary strand 33 pJTI R4 DEST Map of The pJII R4 DEST vector 5583 bp contains the Integrase att R1 and attR2 sites pJTI R4 DEST for the MultiSite Gateway transfer of DNA elements of interest from pDONR entry clones to generate the retargeting expression clone the R4 attB site for site specific integration of the DNA elements into the R4 platform cell line genome and the human EF1a promoter for constitutive expression of resistance to the appropriate selection marker upon successful integration Blasticidin Geneticin or Zeocin depending on the platform vector used The vector sequence of pJTI R4 DEST is available from www invitrogen com or by contacting Technical Support see page 42 pJTI R4 DEST 5583 bp Features of pJTI R4 DEST 5583 nucleotides EF1a bases 66 1244 R4 attB bases 1323 1617 Ampicillin resistance gene ORF bases 1761 2621 pUC origin bases 2766 3439 attR2 recombination site bases 3842 3966 ccdB gene bases 4007 4312 complementary strand Chloramphenicol resistance gene bases 4632 5312 complementary strand attR1 recombination site bases 5421 5545 complementary strand 34 pJTI R4 Int Map of pJTI R4 Int The pJTI R4
52. has occurred you may proceed to retargeting your platform line by cotransfecting with your retargeting expression construct and the pJTI R4 Int vector to generate a stable isogenic cell line expressing your genetic elements of interest This section provides suggestions and helpful hints for generating the retargeting expression construct For generating the retargeting construct using MultiSite Gateway Technology follow the protocol as outlined in the MultiSite Gateway Pro manual 25 0942 supplied with the kit This section does not provide instructions for generating the retargeting construct but provides additional comments and suggestions to help you obtain the best results in multi fragment vector construction Note that the successful assembly of more than 3 fragments is dependent on many variables and following the suggestions below will help maximize the chances of getting the right clone Important For more information on the MultiSite Gateway Technology visit our website at www invitrogen com or contact Technical Support see page 42 MultiSite Two PCR products flanked by specific attB or attBr sites and two MultiSite Gateway Pro Gateway Pro Donor vectors are used in separate BP recombination reactions to TM 2 Fragment generate two entry clones The two entry clones and the pJIT Fast DEST Recombination destination vector are used together in a MultiSite Gateway Pro LR recombination reaction to create your
53. he retargeting expression construct created using the MultiSite Gateway Pro module see the preceding pages is targeted to the RA attP sequences This integration event also positions the constitutive human EFla promoter upstream of the blasticidin neomycin or zeocin resistance gene i e promoterless selection marker thus allowing the selection of transformants that are successfully retargeted using the appropriate selection agent For a map and features of the pJIT R4 Int vector see page 35 The vector sequence of pJTI R4 Int vector is available on our website at www invitrogen com or by contacting Technical Support see page 42 This section provides instructions and guidelines for TM e Cotransfecting your retargeting expression construct and the pJTI R4 Int vector into your R4 platform cell line e Selecting expanding and characterizing your retargeted clones Consult the original references or the supplier of your cell line for optimal method of transfection Methods of transfection include lipid mediated transfection Felgner et al 1989 Felgner amp Ringold 1989 calcium phosphate precipitation Chen amp Okayama 1987 Wigler et al 1977 and electroporation Chu et al 1987 Shigekawa amp Dower 1988 We have obtained the best results for retargeting using high efficiency transfection methods such as microporation or electroporation The following factors are important for successful transfecti
54. his section provides instructions and guidelines for generating the R4 platform line TM For a map and features of the pJIT PhiC31 Int vector and of the each platform vector pJIT Bsd pJTI Neo and pJTI Zeo see pages 30 33 The vector sequences of pJTI PhiC31 Int pJTI Bsd pJTI Neo and pJTI Zeo are available on our website at www invitrogen com or by contacting Technical Support see page 42 You will select stable transformants containing the R4 retargeting sequences by their resistance to Hygromycin B You will not use blasticidin Geneticin G 418 a neomycin analog or Zeocin to select for your platform line as the genes that confer resistance to these agents are promoterless and cannot be expressed at this stage You will use blasticidin Geneticin or Zeocin resistance to select for successfully retargeted clones after the second integration step which will position a constitutive human EF1a promoter upstream of the appropriate resistance gene see the schematic on page 3 TM All pJII platform vectors contain the E coli hygromycin resistance gene HPH Gritz amp Davies 1983 for selection of transfectants with the antibiotic Hygromycin B Palmer et al 1987 When added to cultured mammalian cells Hygromycin B acts as an aminocyclitol to inhibit protein synthesis by disrupting translocation and promoting mistranslation Hygromycin B is available separately from Invitrogen see page
55. ish and at greater than 90 viability prior to transfection e If you are using stem cells in your experiments you must maintain your culture on mitotically inactivated mouse embryonic fibroblast MEF feeder cells or in an appropriate medium conditioned on a MEF feeder layer MEF CM for at least two weeks and as a feeder free culture on MEF CM for at least one passage prior to transfection Make sure to start preparing the feeder layer two days before culturing your stem cells e tiscrucial to allow your cells to recover for at least one day after transfection before you start selection with the appropriate agent If you are using stem cells it is very important to strictly follow the guidelines for culturing your stem cells to keep them undifferentiated Generating the R4 Platform Cell Line Introduction Important Hygromycin B Preparing and Storing Hygromycin B The first step in targeted integration is the generation of the R4 platform line which is accomplished by cotransfecting the pJTI PhiC31 Int vector expressing the PhiC31 integrase and one of the platform vectors pJTI Bsd pJTI Neo or pJ1T Zeo depending on your choice of selection agent for retargeting into your mammalian or stem cells Since the platform vector also contains the Hygromycin resistance gene driven by the thymidine kinase promoter transformants with the desired retargeting sequences are selected in media containing Hygromycin B T
56. it organizations without royalty payment to Invitrogen Invitrogen also understands that Gateway expression clones containing attB1 and attB2 sites may be generated by academic and government researchers for the purpose of scientific research Invitrogen agrees that such clones may be distributed for scientific research by academic and government organizations without royalty payment to Invitrogen Organizations other than academia and government may also distribute such Gateway expression clones for a nominal fee 10 per clone payable to Invitrogen We would ask that such distributors of Gateway entry and expression clones indicate that such clones may be used only for research purposes that such clones incorporate the Gateway Technology and that the purchase of Gateway Clonase from Invitrogen is required for carrying out the Gateway recombinational cloning reaction This should allow researchers to readily identify Gateway containing clones and facilitate their use of this powerful technology in their research Use of Invitrogen s Gateway Technology including Gateway clones for purposes other than scientific research may require a license and questions concerning such commercial use should be directed to Invitrogen s licensing department at 760 603 7200 45 References Ausubel F M Brent R Kingston R E Moore D D Seidman J G Smith J A and Struhl K 1994 Current Protocols in Molec
57. l concentrations of 500 nM Ci2 resazurin and 10 nM SYTOX Green dye Note If the fluorescence intensity of the SYTOX Green dye is too low the final dye concentration can be increased up to 50 nM 6 Incubate the cells at 37 C in an atmosphere of 5 CO for 15 minutes 7 Dilute the cell suspension After the incubation period add 400 uL of the 1X PBS mix gently and keep the samples on ice 8 Analyze the cell sample As soon as possible analyze the stained cells by flow cytometry exciting at 488 nm and measuring the fluorescence emission at 530 nm and 575 nm The population should separate into two groups live cells with a low level of green and a high level of orange fluorescence and necrotic cells with a high level of green fluorescence and a low level of orange fluorescence Confirm the flow cytometry results by viewing the cells with a fluorescence microscope using filters appropriate for fluorescein FITC and tetramethylrhodamine TRITC Freezing Mammalian Cells Introduction Freezing Medium Freezing Protocol for Suspension Cultures We highly recommend that you freeze and bank at least 10 20 vials of cells at each stage of genetic manipulation The cryopreserved cells will supply you with a low passage culture for future genetic manipulations and will ensure that you avoid loss by contamination and minimize genetic changes resulting from continuous culture Cryopreservation will also help prevent aging and transfo
58. m or contact Technical Support see page 42 Jump In TI Gateway Kit is designed to help you genetically engineer stable isogenic mammalian cell lines that express multiple genetic elements of interest using the Jump In Targeted Integration and MultiSite Gateway Technologies Although the kits have been designed to help you construct your cell engineering vectors in the simplest most direct fashion as well as to perform transfection and selection procedures to generate your recombinant cell line expressing your gene s of interest in the most efficient way the use of these products is geared towards users who are familiar with the concepts of the Gateway Technology site specific recombination and culturing mammalian and stem cells If you are unfamiliar with these technologies we recommend that you acquire a working knowledge of the Gateway Technology and methods for maintaining mammalian cell cultures and stem cells Methods General Information Introduction Propagating Jump In TI Gateway System Vectors Important This section provides instructions and guidelines for creating and retargeting your platform cell line using the Jump In TI Gateway System as well as the subsequent selection and expansion It also includes general information on maintaining your mammalian or stem cell culture before and after each transformation However we recommend that you to tailor your cell culture protocols to the specifi
59. mperature at 1 C per minute This can be done by programmable coolers or by placing the vials in an insulated box placed in a 70 C to 90 C freezer then transferring to liquid nitrogen storage Continued on next page 37 Freezing Mammalian Cells continued Freezing Protocol 1 for Adherent Cultures 2 Detach cells from the substrate with the appropriate dissociation agent Detach as gently as possible to minimize damage to the cells Resuspend the detached cells in a complete growth medium and establish the viable cell count Centrifuge at 200 x g for 5 minutes to pellet the cells Using a pipette withdraw the supernatant down to the smallest volume without disturbing the cells Resuspend cells in freezing medium to a concentration of 0 5 x 107 1 x 107 cells ml Aliquot into cryogenic storage vials Place vials on wet ice or in a 4 C refrigerator and start the freezing procedure within 5 minutes Freeze the cells slowly by decreasing the temperature at 1 C per minute This can be done by programmable coolers or by placing the vials in an insulated box placed in a 70 C to 90 C freezer then transferring to liquid nitrogen storage 38 Thawing Mammalian Cells Introduction Centrifugation Method Direct Plating Method Cryopreserved cells are fragile and require gentle handling Thaw cells quickly and plate directly into complete growth medium If cells are particularly sensitive to cryop
60. on e Cells Cells that are 80 90 confluent are ideal for transfection A higher confluency often results in a higher proportion of dead cells in culture Carry out a live dead assay using either FACS LIVE DEAD Cell Vitality Assay Kit see page xi for ordering information or Trypan Blue exclusion counting For more information on using the LIVE DEAD Cell Vitality Assay Kit refer to Assessing Cell Vitality on page 36 e Quality of DNA The quality and the concentration of DNA used play a central role for the efficiency of transfection It is crucial that the DNA is free of endotoxins If using large quantities of DNA we recommend using commercially prepared plasmid DNA For smaller quantities use a commercial kit that delivers pure DNA that is free of endotoxins Do not precipitate DNA with ethanol to concentrate because it reduces efficiency and viability due to the salt contamination e Amount of DNA We generally use 10 ug total plasmid DNA per 2 x 10 8 x 10 cells per transfection but the amount of plasmid DNA may vary depending on the nature of the cell line the transfection efficiency of your cells and the method of transfection used When transfecting your mammalian cell line of choice we recommend that you try a range of plasmid DNA concentrations to optimize transfection conditions for your cell line Continued on next page 23 Retargeting the R4 Platform Cell Line continued If you are transforming stem cells
61. provides instructions for generating Mitomycin C treated mitotically inactivated MEFs Mitomycin C is highly toxic Read and understand the MSDS and handle accordingly Prepare 0 176 w v porcine skin gelatin Sigma Cat no C1890 in sterile distilled water and sterilize by filtration using a 0 2 micron filter Store up to 1 year at 4 C Coat plates for 20 60 minutes at room temperature with 0 1 gelatin in distilled water Prepare 10 ug ml Mitomycin C in MEF medium see below filter sterilize and store at 20 C in the dark until use Mitomycin C can also be kept at 4 C in the dark for up to 2 weeks Mitomycin C is available separately from Sigma St Louis Cat no M4287 Note Used Mitomycin C must be neutralized by addition of 15ml bleach Clorox per 500 ml Mitomycin C solution Swirl to mix incubate for 15 minutes and discard To prepare 500 ml of MEF medium mix the following reagents see pages ix x for ordering information Final Component Volume Concentration D MEM 445 ml 1X FBS 50 ml 10 NEAA 10 mM 5 ml 0 1 mM 2 Mercaptoethanol 1 000X 55 mM 500 pl 55 uM Filter through a 0 22 micron filtration unit to sterilize Pre heat the medium to 37 C before use Hygromycin resistant primary MEFs that are not Mitomycin C treated are available separately from Millipore Cat no PMEF HL or ATCC Cat no SCRC 1045 One vial of cells 5 x 10 6 x 10 cells vial can be used to pl
62. r mammalian cell line of choice we recommend that you try a range of plasmid DNA concentrations to optimize transfection conditions for your cell line If you are transforming stem cells you must maintain your culture on mitotically Important inactivated mouse embryonic fibroblast MEF feeder cells or in an appropriate medium conditioned on a MEF feeder layer MEF CM for at least two weeks and as a feeder free culture on MEF CM for at least one passage prior to transfection Make sure to start preparing the feeder layer two days before culturing your stem cells TM Transfection You may use any of the recommended procedures to co tranfect pJTI PhiC31 Int Procedure and pJTI Bsd pJIT Neo or pJIT Zeo into your cell line of choice Follow the manufacturer s recommendations for transfection Be sure to follow the guidelines outlined below e Remember to include negative controls where either the PhiC31 integrase vector or the platform vector is omitted e Plate the transformed cells in 60 mm culture dishes containing the appropriate medium and allow the cells to recover without selection for at least 24 hours if you have used lipid mediated transfection or 48 72 hours if you have used electroporation or microporation e Wash the cells and provide with fresh medium every day e Each colony recovers at a different rate Monitor morphology and size of the colonies e When your targeted cells have recovered from transfec
63. r sequences Self ligation attR attL 1 Isolate genomic DNA from individual Hygromycin B resistant clones grown to confluency using your preferred method 2 Digest the genomic DNA with a restriction enzyme that does not cut within the pJTI platform vector you have used Stop the restriction digest by heat inactivation If the restriction enzyme cannot be heat inactivated perform a phenol chloroform extraction of the genomic DNA and ethanol precipitate 3 Incubate the restriction fragments with T4 DNA ligase overnight at 16 C under dilute conditions that favor self ligation 4 Extract the DNA from the ligation mixture with phenol chloroform ethanol precipitate the DNA and resuspend in water 5 Electroporate a fraction 25 of the ligated DNA into DH10B T1 electrocompetent E coli see page xii for ordering information using the recommended conditions for the electroporator Plate electroporated cells on LB agar plates containing 100 pg ml ampicillin 7 Isolate the plasmid DNA from resulting colonies and sequence with the following primer to the PhiC31 attB site 5 TCC CGT GCT CAC CGT GAC CAC 3 8 Determine the genomic integration site by matching the sequence read to the database at BLAT www genome ucsc edu cgi bin hgBlat 13 Constructing the Retargeting Expression Vector Introduction Once you have established your R4 platform cell line and confirmed that a single integration event
64. ragment of interest is longer than 3 kb incubate the BP reaction at 16 C overnight instead of 1 hour at room temperature When picking colonies for analysis replica plate them on kanamycin and the drug resistance of the PCR template to reduce the background from template that is inadvertently purified The colonies should only grow on kanamycin Produce clean DNA preparations of the entry clones to use in the LR reaction DNA from minipreps will suffice for the assembly of up to two fragments For assembly of 3 or more fragments midiprep or maxiprep amount and quality DNA is essential Sequence the entry clones with appropriate primers to ensure that the att sites do not have mutations Dilute the DNA to a convenient concentration for the reactions Since the MultiSite Gateway Pro manual recommends 20 femtomoles of the DEST vector and 10 femtomoles of each of the entry vectors per reaction we recommend maintaining a working concentration of 20 fmoles ul for the DEST vector and 10 fmoles yl for each of the entry vectors to allow the addition of 1 pl of each vector to the recombination reaction The vector aliquots should be stored at 20 C While it may be tempting to use a master mix when setting up multiple LR reactions this does not give the best results LR clonase enzyme should always be added at the end Add the DNA first briefly centrifuge the tubes and then add the enzyme to the liquid phase at the bottom
65. reservation centrifuge the cells to remove the cryopreservative DMSO or glycerol and then plate into growth medium We recommend the following procedures adapted from Freshney 1987 for thawing cryopreserved cells Remove the cells from storage and thaw quickly in a 37 C water bath 2 Place 1 or 2 ml of frozen cells in 25 ml of complete growth medium Mix very gently Centrifuge cells at 80 x g for 2 3 minutes and discard the supernatant Gently resuspend the cells in complete growth medium and perform a viable cell count 5 Plate the cells at 2 3 x 10 cells ml Remove the cells from storage and thaw quickly in a 37 C water bath 2 Plate the cells directly using 10 20 ml of complete growth medium per 1 ml of frozen cells Cell inoculum should be at least 3 x 10 cells ml 3 Incubate cells for 12 24 hours and replace the medium with fresh complete growth medium to remove the cryopreservative 39 Generating Mitomycin C Treated MEFs Introduction Preparing Gelatin Coated Plates Preparing Mitomycin C MEF Medium Obtaining MEFs 40 If you are using stem cells in your targeted integration experiments you must maintain your culture on mitotically inactivated mouse embryonic fibroblast MEF feeder cells or in an appropriate medium conditioned on a MEF feeder layer MEF CM for at least two weeks and as a feeder free culture on MEF CM for at least one passage prior to transfection This section
66. rgeting You must passage your platform cell lines at least once without drug selection prior to transfection Stem cell platform lines must be passaged at least once as a feeder free culture on MEF CM and TM Geltrex without drug selection prior to transfection Cells are damaged during harvesting and subsequent handling prior to transfection Avoid damaging cells conditions during harvesting Centrifuge cells at lower speeds 150 200 x g Avoid overexposure to TrypLE trypsin accutase or other dissociation reagents Pipette cells gently Cells remained too long in electroporation cuvette or the Gold Tip Immediately after electroporation microporation transfer cells into pre warmed medium at 37 C to prevent damage Multiple use of Gold Tip if MT 100 MicroPorator is used for transfection Maximum recommend use Gold Tip is between 1 and 3 times because the electric pulses that are applied drastically reduce its quality and impair its physical integrity Low transfection efficiencies Poor optimization of transfection parameters Optimize transfection parameters following electroporator microporator manufacturers recommendations Amount of DNA too low Use the correct amount of DNA for the transfection method of choice following recommended conditions Cell density too low or too high Too low or too high cell densities could drastically reduce the transfection efficiency U
67. rmation if you are using a finite cell line The following freezing protocols have been adapted from Freshney 1987 There are several common media used to freeze cells For serum containing medium the constituents may be as follows e complete medium containing 10 DMSO dimethylsulfoxide or e 50 cell conditioned medium with 50 fresh medium with 10 DMSO If you prefer to cryopreserve your cells in serum free media you should include a protein source to protect the cells from the stress of the freeze thaw process A serum free medium generally has low or no protein but you can still use it as a base for a cryopreservative medium in the following formulations e 50 cell conditioned serum free medium and 50 fresh serum free medium containing 7 5 DMSO e fresh serum free medium containing 7 5 DMSO and 10 cell culture grade BSA 1 Count the number of viable cells to be cryopreserved Cells should be in log phase Centrifuge the cells at 200 400 x g for 5 minutes to pellet Using a pipette remove the supernatant down to the smallest volume without disturbing the cells 4 Resuspend cells in freezing medium to a concentration of 1 x 107 5 x 107 cells ml for serum containing medium or 0 5 x 107 1 x 10 cells ml for serum free medium Aliquot into cryogenic storage vials 5 Place vials on wet ice or in a 4 C refrigerator and start the freezing procedure within 5 minutes 6 Freeze the cells slowly by decreasing the te
68. ro 2 0 Kit for 2 fragment recombination 12537 102 MultiSite Gateway Pro 3 0 Kit for 3 fragment recombination 12537 103 MultiSite Gateway Pro 4 0 Kit for 4 fragment recombination 12537 104 MultiSite Gateway Pro Plus Kit for 2 3 or 4 fragment 12537 100 recombination The table below lists ordering information for competent F coli cells that can be used to propagate your vectors Product Amount Cat no One Shot ccdB Survival 2 T1 Chemically 10 reactions A10460 Competent Cells One Shot Mach1 T1 Chemically Competent 20 x 50 ul C8620 03 Cells One Shot TOP10 Chemically Competent Cells 10 x 50 ul C4040 10 E Shot DH10B T1 Electrocompetent Cells 20 x 25 pl C5100 03 Overview Introduction Components of the Jump In TI Gateway System Note Introduction The Jump In TI Targeted Integration Gateway System combines Invitrogen s MultiSite Gateway Pro cloning and Jump In cell engineering technologies for efficient generation of isogenic mammalian cell lines by enabling irreversible insertion of multiple genetic elements such as promoter reporter pairs at specific locations in the mammalian genome For a detailed explanation of the technology behind the Jump In TI Gateway System see Jump In TI Gateway Cell Engineering Technology on the next page The Jump In TI Gateway System consists of the following components e The Jump In TI Plat
69. s We recommend performing a kill curve as described below to determine the appropriate Blasticidin concentration to use for selecting resistant cells 1 Plate cells at approximately 2576 confluence Prepare a set of 6 plates Allow cells to adhere overnight 2 The next day substitute culture medium with medium containing varying concentrations of Blasticidin e g 0 2 4 6 8 10 ug ml Blasticidin 3 Replenish the selective media every 3 4 days and observe the percentage of surviving cells 4 Determine the appropriate concentration of Blasticidin that kills the cells within 10 14 days after addition of the antibiotic Continued on next page 19 Establishing Sensitivity to Selection Agents continued Geneticin G 418 Preparing and Storing Geneticin Determining Geneticin Sensitivity 20 TM The pJTI Neo platform vector contains the neomycin resistance gene which confers resistance to the antibiotic Geneticin also known as G 418 sulfate Geneticin is available separately from Invitrogen see page xii for ordering information e Geneticin is toxic Do not ingest solutions containing the drug e Wear gloves a laboratory coat and safety glasses or goggles when handling Geneticin and Geneticin containing solutions Follow the instructions provided with Geneticin to prepare your working stock solution Geneticin in powder form should be stored at room temperature and at 4 C as a solu
70. se 1 x 10 cells per microporation or 0 6 x 107 1 0 x 10 cells per electroporation Poor DNA quality Use endotoxin free DNA for all transfections Make sure that the A260 A280 ratio of the DNA is between 1 8 and 2 0 Do not use phenol chloroform extraction or ethanol precipitation Cells are contaminated with Mycoplasma Test cultures for Mycoplasma contamination Start a new culture from a fresh stock 29 Appendix pJTI PhiC31 Int Map of pJTI PhiC31 Int 30 The pJ TII PhiC31 Int vector 6228 bp contains the Streptomyces phage PhiC31 integrase gene under the control of the Cytomegalovirus immediate early promoter CMV The PhiC31 integrase mediates the site specific integration at pseudo attP sites In the Jump In TI Gateway System it is used for site specifically integrating R4 attP retargeting sequences in the genome of the mammalian cell line of choice to create the platform cell line The vector sequence of pJTI PhiC31 Int is available from www invitrogen com or by contacting Technical Support see page 42 pJTI PhiC31 Int 6228 bp Features of pJTI PhiC31 Int 6228 nucleotides T7 promoter bases 1 20 PhiC31Int bases 83 1924 c CMV promoter bases 2113 2636 c pUC origin bases 3121 3794 c Ampicillin resistance gene ORF bases 3981 4841 c SV40 polyA site bases 5777 6139 c c complementary strand pJTI Bsd Map of pJTI Bsd The
71. sensitive 1 2 minutes of incubation is sufficient to detach cells Do not overexpose 8 When cells are sufficiently detached from the flask add 5 ml of MEF medium to each flask rock to disperse and pool cell suspensions from 1 6 flasks into 2 x 50 ml conical tubes 9 Add 15 ml of MEF medium to the first flask to rinse out the cells Rinse the subsequent flask using the same 15 ml MEF medium and pool with cell suspension Discard the flasks 10 Adjust the volume in each tube to 50 ml with MEF medium and centrifuge cells at 200 x g for 4 minutes at room temperature 11 Resuspend cell pellets with MEF medium and pool into one 50 ml tube using a maximum of 12 x T175 flasks of cells per 50 ml tube 12 Centrifuge cells at 200 x g for 4 minutes at room temperature 13 Resuspend the cell pellet in 40 ml of MEF medium using a 10 ml serological pipette and ensuring that the cells are resuspended fully Adjust the volume to 50 ml with MEF medium 14 Centrifuge cells at 200 x g for 4 minutes at room temperature At this stage the cells will have been washed a total of 9 times 6 times before trypsin once at trypsinization and twice post trypsinization 15 Resuspend the cell pellet in 10 ml of MEF medium and then bring to a final volume of 40 ml with MEF medium mixing vigorously before counting cells with trypan blue Mixing is critical to get an accurate cell count 16 Plate MEFs at a density of 3 x 10 cells cm of culture surface
72. structions for generating isogenic stable mammalian cell lines and is supplied with the products listed below Product Cat no Jump In TI Gateway System A10895 Jump In TI Gateway Vector Kit A10896 Jump In TI Platform Kit A10897 Each product contains the following components For a detailed description of the contents of each component see vi viii Cat no Component A10895 A10896 A10897 Jump In TI Platform Kit 4 y Jump In TI Gateway Vector Kit y y MultiSite Gateway Pro Plus Kit y Jump In TI Gateway System Manual y y y The Jump In TI Gateway System and all its components are shipped on dry ice Upon receipt store each component as detailed below All reagents are guaranteed for a minimum of six months if stored properly Item Shipping Storage Vectors Dryice 20 C LR Clonase II Plus Enzyme Mix Dry ice 20 C 6 months 80 C long term BP Clonase II Enzyme Mix Dry ice 20 C 6 months 80 C long term One Shot Mach1 Chemically Competent Dry ice 80 C E coli TM TM Jump In TI Targeted Integration Gateway System Kit is designed to help you genetically engineer stable isogenic cell lines that express multiple genetic elements of interest using the Jump In and MultiSite Gateway Technologies Although the kits have been designed to help you construct your cell engine
73. t in a mixture of 20 ul of Resuspension Buffer and 2 ul of Lysis Solution CellsDirect Resuspension and Lysis Buffers see page xi Incubate the cell suspension at 75 C for 10 minutes no Centrifuge for 1 minute to pellet cell debris Use 3 yl of the cell lysate to set up your PCR TM Successful integration of the pJTI platform vector into the genome of your cell line will result in a PCR product representing the amplified DNA sequence between the R4 attP site and the respective selection marker Bsd Zeo or Neo We highly recommend that you freeze and bank at least 10 20 vials of your R4 platform cells once you have expanded the cell line and confirmed that a single integration event has occurred For instructions on cryopreserving your R4 platform cell line see page 37 Freezing Mammalian Cells in the Appendix Determining Site of Integration Introduction Plasmid Rescue Assay To determine the site of integration in the genome you can perform a plasmid rescue assay and map the site of integration by comparing the recovered sequences to the genomic sequences of your cell line The figure below schematically depicts the plasmid rescue assay where the thin lines represent the genomic DNA from your cell line prior to targeting and the bold lines represent the integrated pJTI platform vector sequences adapted from Chalberg et al 2006 RE cut RE cut Platform Cell Line Genome containing platform vecto
74. th 2 mM hydrogen peroxide for 4 hours at 37 C Untreated cells should be used as a positive control for Ci resazurin staining 1 Preparea1mM stock solution of C12 resazurin Dissolve the contents of the vial of Ciz resazurin Component A in 100 uL of DMSO Component C It may be necessary to agitate the solution in an ultrasonic water bath to fully dissolve the Cy resazurin The Ci resazurin stock solution should be stable for 3 months if stored at x 20 C protected from light Prepare a fresh 50 uM working solution of Ci resazurin by diluting 1 uL of the 1 mM Ci resazurin stock solution in 19 pL of DMSO 2 Prepare a 1 uM working solution of SYTOX Green stain For example dilute 5 uL of the 10 uM SYTOX Green stain stock solution Component B in 45 uL of DMSO Component C The unused portion of this working solution may be stored at x 20 C for up to 1 month 3 Prepare a 1X phosphate buffered saline PBS solution For example for about 20 assays add 2 ml of 10X PBS Component D to 18 ml of deionized water dH2O Pass the 1X PBS through a 0 2 micron filter before use 4 Harvest the cells and dilute as necessary to about 1 x 10 cells ml using the 1X PBS The cells may be washed with 1X PBS if desired 5 Add the dyes to the cell suspension Add 1 uL of the 50 uM Ci resazurin working solution prepared in step 1 and 1 uL of the 1 uM SYTOX Green stain working solution prepared in step 2 to each 100 uL of cell suspension fina
75. tion The stability of Geneticin is guaranteed for six months if stored properly The amount of Geneticin required to be present in culture media to select for resistant cells varies with a number of factors including cell type We recommend that you re evaluate the optimal concentration whenever experimental conditions are altered including use of Geneticin from a different lot Note that Geneticin in powder form has only 75 of the potency of Geneticin available in liquid form 1 Plate or split a confluent plate so the cells will be approximately 25 confluent Prepare a set of 7 plates Allow cells to adhere overnight 2 The next day substitute culture medium with medium containing varying concentrations of Geneticin 0 50 100 250 500 750 and 1000 ug ml Geneticin 3 Replenish the selective media every 3 4 days and observe the percentage of surviving cells 4 Note the percentage of surviving cells at regular intervals to determine the appropriate concentration of Geneticin that kills the cells within 1 2 weeks after addition of Geneticin Continued on next page Establishing Sensitivity to Selection Agents continued Zeocin Preparing and Storing Zeocin Determining Zeocin Sensitivity TM The pJTI Zeo platform vector contains the Sh ble gene Streptoalloteichus hindustanus bleomycin gene the product of which is a 13 7 kDa protein that binds Zeocin and inhibits its DNA strand cleava
76. tion and the colonies are well defined proceed to Selecting Stable Integrants next page Continued on the next page Generating the R4 Platform Cell Line continued Selecting Stable Integrants 10 After your cells have sufficiently recovered from transfection proceed with Hygromycin B selection as described below Use the medium appropriate for your cell line 1 48 to 72 hours after transfection transfer your cells into 100 mm dishes containing fresh medium Split cells such that they are no more than 25 confluent as the selection antibiotics work best at actively dividing cells 2 Incubate the cells at 37 C for 2 3 hours until they have attached sufficiently to the culture dish 3 Remove the medium and add fresh medium containing the appropriate amount of Hygromycin B see page 8 4 Feed the cells with selective medium every 2 3 days until foci can be identified Depending on the cell line colonies will start appearing as early as day 5 of drug selection Mark the colonies and observe them for an additional period of time total of 12 21 days under selection 5 Manually pick single well defined colonies and expand using the appropriate medium under selection for further analysis Screening R4 Platform Cell Line Clones Introduction Southern Blot Analysis What You Should See The PhiC31 integrase catalyzes recombination between two nonidentical sites and lacks a corresponding excisionase enzyme
77. ty to Selection Agents pages 19 22 If you have retargeted stem cells growing on MEF feeders you should also start Hygromycin B selection to prevent overgrowth of your colonies by MEFs 4 Feed the cells with selective medium every 2 3 days until foci can be identified Depending on the cell line colonies will start appearing as early as day 5 of drug selection Mark the colonies and observe them for an additional period of time total of 12 21 days under selection 5 Manually pick single well defined colonies and expand using the appropriate medium under selection for further analysis We recommend that you continue with the Blasticidin Geneticin or Zeocin based selective pressure even after your retargeted clones have been selected and expanded for downstream experiments Continuous selective pressure ensures that expression from your gene s of interest is maintained 25 Screening Retargeted Clones Introduction Preparing Genomic DNA for PCR PCR Analysis Southern Blot Analysis optional 26 Upon retargeting your R4 platform line follow the guidelines below to PCR screen for successful retargeting events using genomic DNA isolated from individual clones Use of nested PCR with primary and secondary reactions is required to eliminate the high background observed with only the primary PCR Pellet 10 000 to 30 000 cells total Wash the cells with 500 pl PBS Centrifuge cells to pellet and remove
78. ular Biology Greene Publishing Associates and Wiley Interscience New York Baron M Reynes J P Stassi D and Tiraby G 1992 A Selectable Bifunctional p Galactosidase Phleomycin resistance Fusion Protein as a Potential Marker for Eukaryotic Cells Gene 114 239 243 Calmels T Parriche M Burand H and Tiraby G 1991 High Efficiency Transformation of Tolypocladium geodes Conidiospores to Phleomycin Resistance Curr Genet 20 309 314 Chen C and Okayama H 1987 High Efficiency Transformation of Mammalian Cells by Plasmid DNA Mol Cell Biol 7 2745 2752 Chu G Hayakawa H and Berg P 1987 Electroporation for the Efficient Transfection of Mammalian Cells with DNA Nucleic Acids Res 15 1311 1326 Drocourt D Calmels T P G Reynes J P Baron M and Tiraby G 1990 Cassettes of the Streptoalloteichus hindustanus ble Gene for Transformation of Lower and Higher Eukaryotes to Phleomycin Resistance Nucleic Acids Res 18 4009 Felgner P L Holm M and Chan H 1989 Cationic Liposome Mediated Transfection Proc West Pharmacol Soc 32 115 121 Felgner P L a and Ringold G M 1989 Cationic Liposome Mediated Transfection Nature 337 387 388 Gritz L and Davies J 1983 Plasmid Encoded Hygromycin B Resistance The Sequence of Hygromycin B Phosphotransferase Gene and its Expression in E coli and S Cerevisiae Gene 25 179 188 Hartley J L Temple G F and Brasch M A 2000 DNA
79. well Continued on next page Generating the R4 Platform Cell Line continued Transfection The following factors are important for successful transfection Considerations e Cells Cells that are 80 90 confluent are ideal for transfection A higher confluency often results in a higher proportion of dead cells in culture Carry out a live dead assay using either FACS LIVE DEAD Cell Vitality Assay Kit see page xi for ordering information or Trypan Blue exclusion counting For more information on how to distinguish metabolically active cells from cells that are dead or injured using the LIVE DEAD Cell Vitality Assay Kit refer to Assessing Cell Vitality on page 36 in the Appendix e Quality of DNA The quality and the concentration of DNA used play a central role for the efficiency of transfection It is crucial that the DNA is free of endotoxins If using large quantities of DNA we recommend using commercially prepared plasmid DNA For smaller quantities use a commercial kit that delivers pure DNA that is free of endotoxins Do not precipitate DNA with ethanol to concentrate because it reduces efficiency and viability due to the salt contamination e Amount of DNA We generally use 10 ug total plasmid DNA per 1 x 10 8 x 10 cells per transfection but the amount of plasmid DNA may vary depending on the nature of the cell line the transfection efficiency of your cells and the method of transfection used When transfecting you
80. you must maintain your culture on mitotically Important inactivated mouse embryonic fibroblast MEF feeder cells or in an appropriate medium conditioned on a MEF feeder layer MEF CM for at least two weeks and as a feeder free culture on MEF CM for at least one passage prior to transfection Make sure to start preparing the feeder layer two days before culturing your stem cells Transfection Use a high efficiency transfection methods such as electroporation or Procedure microporation to co tranfect pJTI R4 Int vector and the retargeting expression construct generated using the MultiSite Gateway Pro module into your R4 platform cells Follow the instructions provided by the manufacturer of the microporation or electroporation apparatus for best results Be sure to follow the guidelines outlined below e Passage your platform cell line at least once without Hygromycin B selection prior to transfection Remember to include negative controls where either the R4 integrase vector or the retargeting expression construct is omitted e Plate the transformed cells in 60 mm culture dishes containing the appropriate medium and allow the cells to recover without selection until the colonies become well defined e Wash the cells and provide with fresh medium every day e Each colony recovers at a different rate Monitor morphology and size of the colonies e When your targeted cells have recovered from transfection and the colonies

Jump-In™ TI™ Gateway® System

Contents

Download Pdf Manuals

Related Search

Related Contents

Jump-In&trade; TI&trade; Gateway&reg; System

Contents

Download Pdf Manuals

Related Search

Related Contents

Jump-In™ TI™ Gateway® System