Retroviral Gene Transfer and Expression User Manual
Contents
1. 2 For infection collect medium from packaging cells and filter medium through a 0 45 um cellulose acetate or polysulfonic low protein binding filter Do not use a nitrocellulose filter because it binds proteins in the retroviral membrane and destroys the virus Optional For VSV G enveloped virus you can concentrate virus as described in Section VII C 3 Add virus to target cells Until you have determined the viral titer use as much virus containing medium as possible for the infection Store remaining viral supernatant at 80 C Clontech Laboratories Inc www clontech com Protocol No PT3132 1 Version No PR842519 Retroviral Gene Transfer and Expression User Manual IX Infecting Target Cells continued Notes Titer will decrease 2 4 fold per freeze thaw cycle Theoptimalfinal concentration of polybrene may needto empirically determined but generally falls within a range of 2 12 ug ml e Excessive exposure to polybrene gt 24 hr can be toxic to cells Alternatively perform infections sequentially 12 hr apart Doing so increases the efficiency of infection but also increases copy number Cellular receptors can be occupied by soluble envelope and or non functional virions Therefore to ensure that cellular receptors will be unoccupied by viral envelope allow cells to rest for a minimum of 12 hr between each infection 4 Add polybrene to a final concentration of 4 8 ug ml Replace medium with fresh med2. Byun J Kim J M Kim S H Yim J Robbins P D amp Kim S 1996 A simple and rapid method for the determination of recombinant retrovirus titer by G418 selection Gene Ther 3 1018 1020 Cashion L M Bare L A Harvey S Trinh O Zhu Y amp Devlin J J 1999 Use of enhanced green fluorescent protein to optimize and quantitate infection of target cells with recombinant retroviruses Biotechniques 26 924 930 Claudio P P Cinti C amp Giordano A 2001 Application of the primer in situ DNA synthesis PRINS technique to titer recombinant virus and evaluation of the efficiency of viral transduc tion Anal Biochem 291 96 101 Coffin J M amp Varmus H E Ed 1996 Retroviruses Cold Spring Harbor Laboratory Press NY Devroe E amp Silver P A 2002 Retrovirus delivered siRNA BMC Biotechnol 2 1 15 Emi N Friedmann T amp Yee J K 1991 Pseudotyped formation of murine leukemia virus with G protein of vesicular stomatitis virus J Virol 65 1202 1207 Freshney R 2000 Culture of Animal Cells Fourth Edition Wiley Liss NY Goff S Traktman P amp Baltimore D 1981 Isolation and properties of Moloney murine leu kemia virus mutants use of a rapid assay for release of virion reverse transcriptase J Virol 38 239 248 Hawley R G Lieu F H L Fong A Z C amp Hawley T S 1994 Versatile retroviral vectors for potential use in gene therapy Gene Ther 1
3. 2 x 10 cells ml www clontech com Clontech Laboratories Inc 13 Retroviral Gene Transfer and Expression User Manual Ill Additional Materials Required Dulbecco s Modified Eagle s Medium high glucose with sodium pyru vate amp glutamine Sigma Cat No D5796 e Fetal bovine serum FBS Note serum need not be heat inactivated 200 mM L Glutamine Sigma Cat No G7513 Solution of 10 000 units ml Penicillin G sodium and 10 000 ug ml Streptomycin sulfate Sigma Cat No P0781 Complete Medium Dulbecco s Modified Eagle s Medium DMEM or Minimum Essential Medium a Modification a MEM supplemented with 100 units ml penicillin G sodium 100 ug ml streptomycin 4 mM Lglutamine 1 mM sodium pyruvate and 10 fetal bovine serum FBS G418 Cat No 631307 Note Make a 10 mg ml active stock solution by dissolving 1 g of powder in approximately 70 ml of complete medium without supplements Filter sterilize and store at 4 C G418 can also be purchased as a premade solution Hygromycin Cat No 631309 Puromycin Cat No 631305 Aminopterin Calbiochem Cat No 454125 Hypoxanthine Calbiochem Cat No 4010 Thymidine Calbiochem Cat No 6060 Zeocin Invitrogen Cat No R250 01 Polybrene Hexadimethrine Bromide Sigma Cat No H9268 Trypsin EDTA Trypsin Sigma Cat No T3924 TNE 50 mM Tris HCI pH 78 130 mM NaCl 1 mM EDTA Dulbecco s phosphate buffered saline DPBS VW
4. Required Typically transfections are done in smaller volumes than culturing For maximal transfection efficiencies we recommend the CalPhos Mammalian Transfection Kit Cat No 631312 For maximal transfec tion efficiency in liposome mediated transfections we recommend Clonfectin Transfection Reagent Cat No 631301 To optimize your transfection protocol you can transfect the host cell line with a non inducible reporter expression vector such as our pLAPSN included in our Retro X System Cat No 631508 or Living Colors Vectors and assay for reporter gene activity After choosing a method of transfection optimize cell density usually 60 80 confluency or 1 2 x 10 cells 60 mm plate the amount and purity of the DNA media conditions and transfection time If a trans fection method is already established in your laboratory proceed with those conditions Keep optimized parameters constant to obtain reproducible results 1 Clone your target gene into a retroviral expression vector or use the provided control vector for control experiments Note Use only high quality plasmid DNA We recommend using a NucleoBond or NucleoSpin Plasmid Kit 2 12 24 hr before transfection plate packaging cells on a 60 mm plate at 60 80 confluency 1 2 x 108 cells 60 mm plate Note Adding 25 uM chloroquine just prior to transfection may increase transfec tion efficiency 2 3 fold Prepare a 25 mM stock of chloroquine in distilled w
5. other general information and precautions http bmbl od nih gov and www niehs nih gov odhsb biosafe nih rdna apr98 pdf Protocol No PT3132 1 www clontech com Clontech Laboratories Inc Version No PR842519 3 Retroviral Gene Transfer and Expression User Manual Il Introduction Retroviral gene transfer is a technique for efficiently introducing stable heritable genetic material into the genome of any dividing cell type Ausubel et al 1995 Coffin et al 1996 This User Manual supports many Clontech packaging cell lines retroviral vectors and retroviral expression systems Retroviral Gene Transfer Technology Current retroviral genetransfertechnology is based on the coordinated design of packaging cell lines and retroviral expression vectors The development of packaging lines cell lines that package recombinant retroviral RNAs into infectious replication incompetent particles created a new level of safety and control Figure 1 Mann et al 1983 Miller amp Buttimore 1986 To develop a packaging cell line the viral gag pol and env genes necessary for particle formation and replication are stably integrated into the genome of the packaging cell line The separate introduction and integration of the structural genes minimizes the chances of producing replication competent virus due to recombination events during cell proliferation Morgenstern amp Land 1990 Miller amp Chen 1996 Retroviral expression vec
6. pantropic packaging cell line Once a packaging cell line is transfected with a retroviral expression vector that contains a packaging signal the viral genomic transcript containing the target gene and selectable marker are packaged into infectious virus within 48 72 hrs Alternatively you can use antibiotic selection to select cells that stably express the integrated vector Stable virus producing cells can be frozen and used in later experiments Virus produced by both transient and stable Clontech Laboratories Inc www clontech com Protocol No PT3132 1 Version No PR842519 Retroviral Gene Transfer and Expression User Manual l Introduction continued Packaging Cell produces viral proteins from stably integrated genes 1 Transfection integration Q ure CK Retroviral QO islas vector expression m stable J 2 Transcription expression Pt gene X Neo DNA wt gene X Neo RNA NP viral Mi proteins 4 Packaging 3 Viral proteins recognize 5 Budding of infectious but 0 replication incompetent virus 6 Collect virus and infect target cells Figure 1 Virus production in packaging cell lines The gag pol and env genes required for viral production are integrated into the packaging cells genome The vector provides the viral packaging signal commonly denoted a target gene and drug resistance marker transfections can infecttargetcells and transmittarg
7. the case of pOCXIX two target genes may be expressed Upon transfection into a packaging cell line O Vectors can transiently express or integrate and stably express a viral genomic transcript containing the CMV immedi ate early promoter gene of interest IRES and antibiotic selection marker Also included in the viral genomic transcript are the necessary viral RNA processing elements including the LTRs packaging signal y and tRNA primer binding site The self inactivating feature of the vectors is provided by a deletion in the 3 LTR enhancer region U3 During reverse transcription Protocol No PT3132 1 www clontech com Clontech Laboratories Inc Version No PR842519 9 Retroviral Gene Transfer and Expression User Manual Il Introduction continued of the retroviral transcript in the infected cell the inactivated 3 LTR is copied and replaces the 5 LTR resulting in inactivation of the 5 LTR promoter CMV The gene of interest and antibiotic resistance gene are expressed from an internal CMV promoter and co translated via the internal ribosome entry site IRES as a bicistronic message in mammalian cells Jackson et al 1990 Jang et al 1988 Creator Compatibility for Diverse Gene Expression Studies Our retroviral systems are fully compatible with the Creator Gene Cloning andExpression System This system uses Cre loxP recombination to transfer ageneofinterestdirectly from a single donor vector into numerous accept
8. 01 High efficiencies of gene transfer with immobilized recombinant retrovirus kinetics and optimization Biotechnol Prog 17 4 587 596 H Cocultivation of target cells and packaging cells Allows targets to be continuously in contact with freshly produced viral supernatant Casal M L amp Wolfe J H 1997 Amphotropic and ecotropic retroviral vector viruses transduce midgestational murine fetal liver cells in a dual chambered cocultivation system Gene Ther 4 1 39 44 Germeraad W T Asami N Fujimoto S Mazda O amp Katsura Y 1994 Efficient retrovirus mediated gene transduction into murine hematopoietic stem cells and long lasting expression using a transwell coculture system Blood 84 3 780 788 I Use of cationic liposomes Enhance virus to cell fusion Kaneko Y amp Tsukamoto A 1996 Cationic liposomes enhance retrovirus mediated multinucleated cell formation and retroviral transduction Cancer Lett 105 1 39 44 Porter C D Lukacs K V Box G Takeuchi Y amp Collins M K 1998 Cationic liposomes enhance the rate of transduction by a recombinant retroviral vector in vitro and in vivo J Virol 72 6 4832 4840 J Use of histone deacetylase inhibitors to increase titer Relieves repression of viral expression by hyperacetylation of histones Chen W Y Bailey E C McCune S L Dong J Y amp Townes T M 1997 Reactivation of silenced virally transduced genes by inhibitors of histon
9. 1 www clontech com Clontech Laboratories Inc Version No PR842519 7 Retroviral Gene Transfer and Expression User Manual Introduction continued TABLE II PACKAGING CELL LINES Retropack PT67 EcoPack2 AmphoPack 293 293 Env specific cells mammalian mammalian non mammalian Envelope 10A1 gap70 4070A VSV G Env specific Receptors GALV Pit1 mCAT 1 RAM1 phosphatidylserine RAM Pit2 phosphatidyl inos itol amp Gus gangli oside Markers gag pol Bleo Bleo DHFR env BHER Hyg Puro production of high titer amphotropic retrovirus Figure 2 AmphoPack 293 cells can also be used to produce high titer retrovirus stably Bleomy cin and puromycin resistance genes were used to separately introduce the viral gag pol and env genes Therefore the popular neomycin and hygromycin selection markers can be used to develop clones that stably produce high titer virus Virus produced by AmphoPack 293 cells express an amphotropic envelope 4070A and thus can infect a broad range of mammalian cell types Table l e GP2 293 Packaging Cell Line The Pantropic Retroviral Expression System Cat No 631512 features GP2 293 a HEK 293 based packaging cell line that stably expresses the viral gag and pol genes To produce infectious virus cotransfect GP2 293 with a retroviral expression vector and pVSV G a plasmid that expresses VSV G from the CMV promoter Yee et al 1994 The VSV G envelope must be cotransfected with t
10. 136 138 Hemann M T Fridman J S Zilfou J T Hernando E Paddison P J Cordon Cardo C Hannon G J amp Lowe S W 2003 An epi allelic series of p53 hypomorphs created by stable RNAi produces distinct tumor phenotypes in vivo Nat Genet 33 3 396 400 Hilberg F 1987 Functional analysis of a retroviral host range mutant altered long terminal repeat sequences allow expression in embryonal carcinoma cells Proc Natl Acad Sci USA 84 5232 5236 Higashikawa F amp Chang L 2001 Kinetic Analysis of stability of simple and complex retroviral vectors Virology 280 124 131 Jackson R J Howell M T amp Kaminski A 1990 The novel mechanism of initiation of picor navirus RNA translation Trends Biochem Sci 15 477 483 Jang S K Krausslich H G Nicklin M J Duke G M Palmenberg A C amp Wimmer E 1988 A segment of the 5 nontranslated region of encephalomyocarditis virus RNA directs internal entry of ribosomes during in vitro translation J Virol 62 2636 2643 Kozak M 1987 At least six nucleotides proceeding the AUG initiator codon enhances transla tion in mammalian cells J Mol Biol 196 947 950 Protocol No PT3132 1 www clontech com Clontech Laboratories Inc Version No PR842519 3 Retroviral Gene Transfer and Expression User Manual XI References continued Kwon Y J Hung G Anderson W F Peng C A amp Yu H 2003 Determination of infectious retrovirus con
11. 200 Bahnson A B Dunigan J T Baysal B E Mohney T Atchison R W Nimgaonkar M T Ball E D amp Barranger J A 1995 Centrifugal enhancement of retroviral medi ated gene transfer J Virol Methods 54 131 143 Precipitation to increase titer concentration Pham L Ye H Cosset F L Russell S J amp Peng K W 2001 Concentration of viral vectors by co precipitation with calcium phosphate J Gene Med 3 2 188 194 Darling D Hughes C Galea Lauri J Gaken J Trayner D Kuiper M amp Farzaneh F 2000 Low speed centrifugation of retroviral vectors absorbed to a particulate substrate a highly effective means of enhancing retroviral titre Gene Ther 7 11 914 923 Hughes C Galea Lauri J Farzaneh F amp Darling D 2001 Streptavidin paramagnetic Clontech Laboratories Inc www clontech com Protocol No PT3132 1 Version No PR842519 Retroviral Gene Transfer and Expression User Manual Appendix C Additional Viral Infection Methods continued particles provide a choice of three affinity based capture and magnetic concentration strategies for retroviral vectors Mol Ther 3 4 623 630 D Precipitation during transduction facilitates greater contact between the target cells and virions Le Doux J M Landazuri N Yarmush M L amp Morgan J R 2001 Complexation of retrovirus with cationic and anionic polymers increases the efficiency of gene transfer Hum G
12. Laboratories Inc Version No PR842519 41
13. Plasmid may rearrange due to presence of LTR s Switch to alternate E coli strain for unstable DNA sequences Improper thawing procedures follow thawing procedures in Section VI B Improper culture medium all packaging cell lines will grow in DMEM 10 FBS Improper tissue culture plasticware use collagen l coated plates to aid adherence during initial seeding Improperculturemedium all packagingcelllineswill grow in DMEM 10 FBS Improper incubator conditions grow cells at 37 C in humidified incubator with 5 10 CO Improperculture medium all packaging cell lines will grow in DMEM 10 FBS Improper tissue culture plasticware use collagen l coated plates to aid adherence during initial seeding Subclone parental cell line Incorrect amount of antibiotic we do not recommend reselecting for packaging function Protocol No PT3132 1 Version No PR842519 www clontech com Retroviral Gene Transfer and Expression User Manual X Troubleshooting Guide continued C Virus Production Poor Transfection efficiency Low titer 105 cfu ml Protocol No PT3132 1 Version No PR842519 Cells are overly confluent plate fewer cells 60 80 confluency 1 2 x 10 cells 60mm Transfection is toxic to cells Optimize DNA and transfection reagent amounts and exposure time Assaying for positive cells too early wait 48 hr after transfection for maximal gene expression to determine efficiency Poortrans
14. R Cat No 82020 066 Cell Freezing Medium Sigma Cat No C6164 or DMSO Sigma Cat No D2650 Tissue culture plates and flasks BD Biocoat Collagen Type I 12 well plates BD Biosciences Cat Nos 354500 amp 356500 Cloning cylinders PGC Scientific Cat No 62 6150 40 45 NIH 3T3 cells ATCC Cat No CRL1658 CalPhos Mammalian Transfection Kit Cat No 631312 CLONfectin Transfection Reagent Cat No 631301 Chloroquine Sigma Cat No C6628 Clontech Laboratories Inc www clontech com Protocol No PT3132 1 14 Version No PR842519 Retroviral Gene Transfer and Expression User Manual IV Safety amp Handling of Retroviruses The protocols in this User Manual require producing handling and storing infectious retrovirus Athorough understanding of safe laboratory practices and potential retroviral hazards is essential MMLV does not naturally infect human cells however viruses packaged from the MMLV based vectors described here are capable of infecting hu man cells if packaged in a cell line with the proper tropism This statement is also true for PCMV based vectors The viral supernatants produced by these retroviral systems could depending on your retroviral insert contain potentially hazardous recombinant virus For these reasons exercise due caution when producing and handling recombinant retrovirus The user is strongly advised not to create retrovi ruses capable of expressing kn
15. R Primer 20 uM e 100 ul 3 pLNCX Seq PCR Primer 20 uM MSCV Retroviral Expression System Cat No 634401 e 1 ml RetroPack PT67 Cell Line 2 x 106cells ml 20 ug pMSCVneo Retroviral Vector 0 5 ug ul 20 ug pMSCVhyg Retroviral Vector 0 5 ug ul 20 ug pMSCVpuro Retroviral Vector 0 5 ug ul e 100 ul 5 pMSCV Primer 20 uM e 100 ul 3 pMSCV Primer 20 uM Pantropic Retroviral Expression System Cat No 631512 e 1 ml GP2 293 Packaging Cell Line 2 x 108cells ml e 1 ml GP 293 Luc Packaging Cell Line 2 x 108cells ml 20 ug pLNHX Vector 0 5 ug ul 20 ug pLXRN Vector 0 5 ug ul 20 ug pLLRN Control Vector 0 5 ug ul 20 ug pVSV G Vector 0 5 ug ul 12 Version No PR842519 Retroviral Gene Transfer and Expression User Manual Il List of Components continued Retro X Universal Packaging System Cat No 631530 1 20 20 20 20 20 ml Hg Hg Hg Hg Hg GP2 293 Packaging Cell Line 2 x 108cells ml p10A1 Vector 0 5 ug ul pAmpho Vector 0 5 ug ul pEco Vector 0 5 ug ul pVSV G Vector 0 5 ug ul pOCLIN Control Vector 0 5 ug ul RetroPack PT67 Cell Line Cat No 631510 1 ml RetroPack PT67 Cell Line 2 x 10 cells ml EcoPack2 293 Cell Line Cat No 631507 1 ml EcoPack2 293 Cell Line 2 x 10 cells ml AmphoPack 293 Cell Line Cat No 631505 1 Protocol No PT3132 1 Version No PR842519 ml AmphoPack 293 Cell Line
16. T S G don o N Retroviral Gene Transfer and Expression User Manual Clontech United States Canada 800 662 2566 Asia Pacific 1 650 919 7300 Europe 33 0 1 3904 6880 Japan 81 0 77 543 6116 Clontech Laboratories Inc PT31 32 1 PR84251 9 ATakara Bio Compan t 1290 Terra Bella Ave Published 15 April 2008 Mountain View CA 94043 Technical Support US E mail tech clontech com www clontech com Retroviral Gene Transfer and Expression User Manual Table of Contents l Il Ill IV V VI VII VIII X XI Introduction List of Components Additional Materials Required Safety amp Handling of Retroviruses Plasmid Manipulations A Propagating Plasmids B Generating Expression Vectors Culturing Packaging Cell Lines A General Considerations B Starting Cells from Frozen Stocks C Maintaining Packaging Cell Lines D Freezing Packaging Cell Lines Virus Production A Transfecting Retroviral Vectors B Selecting Stable Virus Producing Cell Lines C Concentrating Virus D Producing Virus from Stable Packaging Cell Clone PT67 Storage of Viral Stocks Determining Viral Titer A General Considerations B Procedure for Determining Viral Titer C Alternative Methods Infecting Target Cells A General Considerations B Infecting Target Cells Troubleshooting Guide References Appendix A Culture Plate Conversions Appendix B Titration of Antibiotic Stocks Kil
17. T67 Cell Line is covered under U S Patent No 5 766 945 which has been assigned to the Fred Hutchinson Cancer Research Center Retroviral vectors are sold under license from Fred Hutchinson Cancer Research Center Rights to use this product are limited to research only No other rights are conveyed Inquiry into the availability of a license to broader rights or the use of this product for commercial purposes should be directed to Fred Hutchinson Cancer Research Center Technology Transfer Office 1100 Fairview Avenue North J6 200 Seattle WA 98109 Purchase of this product does not grant rights to 1 offer the materials or any derivatives thereof for resale 2 to distribute or transfer the materials or any derivatives thereof to third parties Use of the MSCV Retroviral Expression System is exclusively licensed from Robert G Haw ley Ph D Academic research institutions are granted an automatic license with the purchase of this product to use the MSCV Retroviral Expression System only for internal academic research purposes This limitation expressly excludes the right to sell or otherwise transfer the MSCV system or its component parts to third parties All other users and purchasers are required to obtain a license from Clontech prior to purchasing these reagents or using them for any purpose For information on licensing please contact a licensing representative by phone at 650 919 7320 or by e mail at licensing 9 clontech com Use of the P
18. al 1997 and PCR applied to viral supernatants Quinn amp Trevor 1997 Morgan et al 1990 Reverse transcriptase ac tivity has also been used Goff et al 1981 Some have used protein production encoded by the retroviral transgene from packaging cells as a method for screening high titer clones but this method is flawed because protein production does not correlate with the number of infectious virions Tasaki et al 1997 Infecting Target Cells General Considerations The following protocols are general recommendations for infecting adherent cells such as NIH 3T3 or HeLa Use them as a starting point for determining optimal conditions for your experiments If these protocols do not work for your cell type please refer to Appendix C for alternative infection methods Important Multiple rounds of infection can improve your results by increasing the number of infected cells as well as increasing the copy number per cell Virus produced with the VSV G envelope can be concentrated by ul tracentrifugation to titers of up to 10 cfu ml See Section VII C Infecting Target Cells 1 Plate the target cells 12 18 hr before infection at a cell density of 1 2 x 10 per 60 mm plate If you will be using infected cells for a biological assay ensure that the control cells are treated with an insert free virus under identical conditions Note The viral pre integration complex enters the nuclei of actively dividing cells only
19. all be used with our various packaging cell lines For more detailed descriptions of our vectors and sequence information visit our www clontech com and navigate to the vector information page All vectors contain the extended retroviral packaging signal Y which promotes high titer virus production With the exception ofthe expression vectors in the MSCV Retroviral Expres sion System Cat No 634401 all vectors are derived from Moloney murine leukemia virus MMLV Each vector contains a different antibiotic resistance marker neomycin hygromycin or puromycin allowing you to choose the cloning vector appropriate for the desired selection method The MSCV Vectors contain a specifically designed long terminal repeat LTR from the murine stem cell PCMV virus PCMV stands for PCC4 cell passaged myeloproliferative sarcoma virus Hilberg etal 1987 Hawley etal 1994 This LTR differs from the MMLV LTR by several point mutations and a deletion These changes enhance transcriptional activation and decrease transcrip tional suppression in embryonic stem and embryonal carcinoma cells As a result the LTR drives high level constitutive expression of a target gene in stem cells and other mammalian cell lines Hawley et al 1994 The Retro X O Vectors are self inactivating bicistronic expression vec tors designed to express a target gene along with an antibiotic selection marker without the risk of promoter interference from the 5 LTR In
20. amination of solid and liquid waste e Use unrecirculated exhaust air e Stock chemical disinfectants for spills Protocol No PT3132 1 www clontech com Clontech Laboratories Inc Version No PR842519 Retroviral Gene Transfer and Expression User Manual V Plasmid Manipulations A Propagating Plasmids 1 2 To ensure that you have a renewable source of DNA transform each plasmid into a suitable E coli host strain e g DH5a Purify plasmids with a NucleoBond or NucleoSpin Plasmid Kit Alternatively isolate plasmids by banding on a CsCl gradient Sambrook amp Russell 2001 B Generating Expression Vectors Use standard molecular biology techniques to transfer your target gene into an expression vector Sambrook amp Russell 2001 1 AR Purify your gene fragment by any standard method The cDNA or gene fragment must contain an ATG initiation codon Adding a Kozak consensus ribosome binding site may improve expression levels in mammalian systems Kozak 1987 Please note that all sequences placed into a retroviral vector must be compatible with the retroviral life cycle and allow complete transcription ofthe full length viral genome Sequences such as poly A signals must not be included Coffin et a 1996 You can generate the fragment using compatible restriction sites that are on either side of the gene and in the cloning vector If no such sites are present use PCR to incorporate suitable restrictio
21. and 10A1 envelope protein For example virus produced from RetroPack PT67 cells cannot efficiently infect AmphoPack 293 cells and vice versa Virus packaged in GP2 293 cells can be used to infect any other cell line depending on the envelope pVSV G pEco pAmpho or p10A1 that was cotransfected with the expression vector Virus produced by EcoPack2 293 cells can only infect mouse and rat cells such as RetroPack PT67 cells Protocol No PT3132 1 www clontech com Clontech Laboratories Inc Version No PR842519 21 Retroviral Gene Transfer and Expression User Manual VII Virus Production continued B Selecting Stable Virus Producing Cell Lines 1 Prior to using antibiotics to establish stable cell lines you must titrate antibiotic stocks to determine the optimal concentration for selection see Appendix B This procedure is commonly called a kill curve 2 Plate transfected packaging cells in selection medium 24 36 hr after transfection 3 Culture cells for one week with the appropriate antibiotic 4 Isolate large healthy colonies andtransferthem to individual plates or wells Note We generally isolate clones using cloning cylinders or cloning disks The se lected cell populations usually produce titers of 10 cfu ml If you require higher titer clones pick individual clones for propagation Determine viral titer as described in Section VIII You must screen 20 50 clones to isolate a clone of acceptably high
22. antropic Retroviral Expression System and the Retro X Universal Packaging System are licensed from Pangenix and are covered under U S Patent Nos 5 512 421 and 5 670 354 Rights to use this product are limited to nonhuman research only and use of the patented technology in domestic ungulates is expressly prohibited No other rights are con veyed Inquiry into the availability of a license for commercial purposes should be directed to Jane C Burns M D Pangenix 6505 EI Camino delTeatro La Jolla CA 92037 VSV G is licensed from Pangenix and its use is covered under U S Patent Nos 5 512 421 and 5 670 354 Rights to use this product are limited to nonhuman research only and use of the patented technology in domestic ungulates is expressly prohibited No other rights are conveyed Inquiry into the availability of a license for commercial purposes should be directed to Jane C Burns M D Pangenix 6505 EI Camino delTeatro La Jolla CA 92037 DsRed Monomer This product is covered under U S Patent No 7 250 298 Polybrene is a registered trademark of Abbott Laboratories Inc NucleoSpin amp and NucleoBond are registered trademarks of MACHEREY NAGEL GmbH amp Co KG Zeocin is a trademark of CAYLA Clontech the Clontech Logo andall othertrademarks arethe property of Clontech Laboratories Inc unless noted otherwise Clontech is aTakara Bio Company 2008 Clontech Laboratories Inc Protocol No PT3132 1 www clontech com Clontech
23. ater and filter sterilize 1 2 hr before transfection replace medium with medium containing chloroquine Pear et al 1993 3 Transfect each 60 mm plate with the following amount of plasmid DNA For RetroPack PT67 EcoPack 2 293 amp AmphoPack 293 5 10 ug of plasmid DNA For GP2 293 5 ug of expression vector and 5 ug envelope vector Notes When using GP2 293 cells envelope vector must be cotransfected e When using a CaPO based transfection method the final volume of transfection mixture should not exceed 0 5 ml for a 60 mm plate or 1 ml for a 100 mm plate More than 1 ml of CaPO precipitants can be toxic to cells Add the transfection solution to the medium and evenly distribute the solution on the cells If toxicity is observed perform transfection with 0 5 ml of the transfection mix e 6 8hr after transfection you may perform glycerol shock treatment to increase the uptake of DNA Freshney 2000 Clontech Laboratories Inc www clontech com Protocol No PT3132 1 Version No PR842519 Retroviral Gene Transfer and Expression User Manual VII Virus Production continued 4 For RetroPack PT67 cells aspirate culture medium 10 24 hr after transfection Wash RetroPack PT67 cells twice with PBS and add 3 ml of complete medium Proceed to Section VII B 5 For HEK 293 based cell lines aspirate culture medium 8 10 hr after transfection and add 3 ml of complete medium 6 Incubate the culture for an ad
24. centration from colony forming assay with quantitative analysis J Virol 77 5712 5720 Mann R Mulligan R C amp Baltimore D 1983 Construction of a retrovirus packaging mutant and its use to produce helper free defective retrovirus Cell 33 153 159 Miller A D 1996 Cell surface receptors for retroviruses and implications for gene transfer Proc Natl Acad Sci USA 93 11407 11413 Miller A D amp Buttimore C 1986 Redesign of retrovirus packaging cell lines to avoid recom bination leading to helper virus production Mol Cell Biol 6 8 2895 2902 Miller A D amp Chen F 1996 Retrovirus packaging cells based on 10A1 murine leukemia virus for production of vectors that use multiple receptors for cell entry J Virol 70 8 5564 5571 Miller D G amp Miller A D 1994 A family of retroviruses that utilize related phosphate trans porters for cell entry J Virol 68 8270 8276 Miyao Y Shimizu K Tamura M Yamada M Tamura K Nakahira K Kuriyama S Hayakawa T amp IKenaka K 1995 A simplified general method for determination of recombinant retrovirus titers Cell Struct Funct 20 177 183 Morgan R A Cornetta K amp Anderson W F 1990 Applications of the polymerase chain re action in retroviral mediated gene tranfer and the analysis fo gene marked human TIL cells Hum Gene Ther 1 135 149 Morgenstern J P amp Land H 1990 Advanced mammalian gene transfer high titer ret
25. ction To produce high titer virus tran siently transfect a retroviral expression vector into an HEK 293 based packaging cell line After 48 72 hours collect virus and determine the viral titer or infect a target cell line Alternatively you can use antibiotic selection to develop clones that stably produce high titer retrovirus In addition Clontech offers a variety of stable packaging cell lines Table II provides a detailed overview of each cell line e RetroPack PT67 Cell Line The RetroPack PT67 Cell Line Cat No 631510 is derived from a mouse fibroblast NIH 3T3 cell line designed for stably producing high titer retro virus RetroPack PT67 cells package virus with a dualtropic or polytropic envelope 10A1 that recognizes receptors on mouse rat human ham ster mink cat dog and monkey cells Virus produced by these cells can enter target cells via two surface molecules the amphotropic retrovirus receptor RAM1 Pit2 and the GALV Pit1 receptor Two viral receptors means that if one receptor is not abundantly expressed by a given spe Clontech Laboratories Inc www clontech com Protocol No PT3132 1 6 Version No PR842519 Retroviral Gene Transfer and Expression User Manual Introduction continued TABLE I HOST RANGE OF PACKAGING CELL LINES EXPRESSING DIFFERENT ENVELOPES cene opes ER See Table Il Packaging Cell Lines for a description of the envelope proteins This listing of the most commo
26. days For selecting stable transfectants use a plating density that allows the cells to reach 80 confluency before massive cell death begins at about day 5 This is the cell density at which cells should be plated for selection of stable transfectants Protocol No PT3132 1 www clontech com Clontech Laboratories Inc Version No PR842519 35 Retroviral Gene Transfer and Expression User Manual Appendix C Additional Viral Infection Methods These references are provided for fine tuning your transduction protocols to achieve the desired infection frequency in target cells This list is not in tended to be comprehensive These protocols will work for a wide range of cell types however you must determine which works best for your targets While each technique can provide modest increases in efficiency they may be combined to create an additive effect For ease of analysis we recommend our retroviral vectors that express our Living Colors fluorescent proteins for detection and quantitation of gene transfer efficiency during testing A Transduction of cells at 32 C Decrease in temperature increases viral half life during transduction Bunnell B A Muul L M Donahue R E Blaese R M Morgan R A 1995 High efficiency retroviral mediated gene transfer into human and nonhuman primate pe ripheral blood lymphocytes Proc Natl Acad Sci USA 92 17 7739 7743 Zhou P Lee J Moore P Brasky K M 2001 High e
27. ditional 48 72 hr to allow viral titer to increase The viral titer reaches a maximum 48 hr after transfection and is generally at least 3096 of the maximum beyond 72 hr after transfection Alternative Method Infecting packaging cells Ping Pong This method can be used to deliver the viral construct to the packag ing cell line an objective that can be accomplished by transfection electroporation or even infection Note Retro X Q vectors can only be delivered by transfection This method can also eliminate the need for selecting individual clones when making stable packaging cell lines Parente amp Wolfe 1996 Alternatively infect the packaging cells with virus obtained from another packaging cell line Table II details the appropriate packaging cell lines to use for infection A protocol for infection follows in Section VIII This approach produces high titer virus for several reasons More cells acquire the construct and copy number is higher and more consistent 1 2 copies per cell per single round of infection depending upon titer of virus stock Virus producing clones derived from transduced cells are more stable than those derived from transfected cells Parente amp Wolfe 1996 Allows the host range of a vector to be changed Important Notes This method requires previously transfected virus producing packaging cells You cannot infect cells that are already expressing the same or similar eg Ampho
28. e deacetylase Proc Natl Acad Sci USA 94 5798 5803 Tobias C A Kim D amp Fischer l 2000 Improved recombinant retroviral titers utilizing trichostatin A Biotechniques 29 884 890 Clontech Laboratories Inc www clontech com Protocol No PT3132 1 Version No PR842519 Retroviral Gene Transfer and Expression User Manual Notes Protocol No PT3132 1 www clontech com Clontech Laboratories Inc Version No PR842519 39 Retroviral Gene Transfer and Expression User Manual Notes Clontech Laboratories Inc www clontech com Protocol No PT3132 1 Version No PR842519 Retroviral Gene Transfer and Expression User Manual Notice to Purchaser Notice to Purchaser Clontech products areto be used for research purposes only They may not be usedforany other purpose including but not limited to use in drugs in vitro diagnostic purposes therapeutics or in humans Clontech products may not be transferred to third parties resold modified for resale or used to manufacture commercial products or to provide a service to third parties without written approval of Clontech Laboratories Inc For Research Use Only Not for Use in Humans Use of the Retro X Universal Packaging System AmphoPack 293 and EcoPack 2 293 are covered by U S Patent No 5 858 40 and are limited to use solely for research purpos es Please contact GBP IP LLC for a license to use these products for commercial purposes The RetroPack P
29. ee Coffin amp Varmus 1996 or Ausubel et al 1995 Figure 3 provides an overview of methods for producing high titer virus using RetroPack PT67 EcoPack2 293 Amphopack 293 and GP2 293 cells Transient Virus Production Stable Virus Production RetroPack PT67 1 Transfect with retroviral vector 2 Select stable clones 3 Determine viral titer 4 Infect target cells EcoPack2 293 amp AmphoPack 293 1 Transfect with retroviral vector 2 Determine viral titer or 2 Select stable clones 3 Infect target cells 3 Determine viral titer 4 Infect target cells GP2 293 1 Cotransfect with retroviral vector or 1 Transfect with retroviral vector amp envelope vector pVSV G pEco omit envelope vector pAmpho or p10A1 2 Select stable clones 2 Determine viral titer 3 Before each infection transiently 3 Concentrate virus transfect with envelope vector optional for VSV G 4 Concentrate virus optional 4 Infect target cells 5 Determine viral titer 6 Infect target cells Figure 3 Overview of producing infectious retrovirus Protocol No PT3132 1 www clontech com Clontech Laboratories Inc Version No PR842519 19 Retroviral Gene Transfer and Expression User Manual VII Virus Production continued A Transfecting Retroviral Vectors Transfect by any standard method We routinely use 60 mm plates for culturing packaging cell lines See Additional Materials
30. ell culture using sterile technique in a suitable hood For those requiring more information on mammalian cell culture we recommend the fol lowing general references e Culture of Animal Cells Fourth Edition ed by R I Freshney 2000 Wiley Liss e Current Protocols in Molecular Biology ed by F M Ausubel et al 1995 Wiley amp Sons Note Frozen cells should be cultured immediately upon receipt or as soon thereafter as possible Increased loss of viability may occur after shipping if culturing is delayed 1 Transfer the vial of frozen cells from liquid N to a 37 C water bath until just thawed To prevent osmotic shock and to maximize cell survival perform the following a Rinse the outside of the tube with 7096 ethanol b Add 1 ml complete medium prewarmed to 37 C to tube Transfer mixture to a 15 ml tube c Add 5 ml complete medium and mix gently Repeat The final volume should be 12 ml d Centrifuge at 250 x g for 10 min e Remove supernatant 2 Gently resuspendcells in 10 ml complete medium DMEM or Mini mum Essential Medium a Modification a MEM supplemented with 100 units ml penicillin G sodium 100 ug ml streptomycin 4 mM L glutamine 1 mM sodium pyruvate and 10 fetal bovine serum 3 Incubate cells at 37 C with 5 CO Protocol No PT3132 1 www clontech com Clontech Laboratories Inc Version No PR842519 17 Retroviral Gene Transfer and Expression User Manual VI Culturing Packaging Cell L
31. eneTher 12 13 1611 1621 Morling F J amp Russell S J 1995 Enhanced transduction efficiency of retroviral vec tors coprecipitated with calcium phosphate Gene Ther 2 7 504 508 Hennemann B Chuo J Y Schley P D Lambie K Humphries R K amp Eaves C J 2000 High efficiency retroviral transduction of mammalian cells on positively charged surfaces Hum GeneTher 11 1 43 51 E Increase transduction rate by phosphate depletion results in up regulation of GLVR 1 and GLVR 2 RAM1 receptors for ampho tropic or 10A1 pseudotyped virus Bunnell B A Muul L M Donahue R E Blaese R M amp Morgan R A 1995 High efficiency retroviral mediated gene transfer into human and nonhuman primate pe ripheral blood lymphocytes Proc Natl Acad Sci USA 92 17 7739 7743 Zhou P Lee J Moore P Brasky K M 2001 High efficiency gene transfer into rhesus macaque primary T lymphocytes by combining 32 degrees C centrifugation and CH 296 coated plates effect of genetransfer protocol onT cell homing receptor expression Hum GeneTher 12 15 1843 1855 F Flow through transduction concentrating cells and virus together in small culture systems Pan D Shankar R Stroncek D F amp Whitley C B 1999 Combined ultrafiltration transduction in a hollow fiber bioreactor facilitates retrovirus mediated gene transfer into peripheral blood lymphocytes from patients with mucopolysaccharidosis type Il H
32. et genes however itcan not replicate within target cells because the viral structural genes are absent The Retro X Universal Packaging System Cat No 631530 is a transient packaging system that allows you to select the envelope according to the tropism needed for your experiments It includes the GP2 293 cell line which has the viral gag and pol genes incorporated in its genome The remaining portion of the packaging function the viral env gene must be cotransfected with the retroviral expression vector bearing the gene of interest The kit includes vectors that encode ecotropic amphotropic dualtropic 10A1 and pantropic VSV G envelope proteins This allows you to cater the tropism or host range of the packaged virus to your needs by determining which envelope protein is used Protocol No PT3132 1 www clontech com Clontech Laboratories Inc Version No PR842519 Retroviral Gene Transfer and Expression User Manual l Introduction continued Transient Production Stable Production Plate packaging cells Plate packaging cells Q Transfect Transfect Q Expression 8 10 hr 24 hr Expression Vector Vector Target Cell Infection Plate target cells 48 72 hr 2 weeks ES SO C I OgooddSoc D Collect virus V Dolla eS c ce Screen for high titer clones 2 weeks Select cells and analyze Expand amp collect virus 1 week Figure 2 Overview of transient and stable virus produ
33. fection efficiency optimize transfection Concentrate virus if using VSV G Truncated viral RNA check sequence for presence of poly A between LTRs Virus harvested too early harvest virus 48 72 hr after transfection Vector too large The limit of packaging function is 8 3 kb from LTR to LTR Concentrate virus for large vectors or reduce size of inserts Low virus production from cell population PT67 Pick and screen for stable higher titer clones No polybrene added during titration add polybrene 4 8 ug ml to viral supernatant Virus exposed to multiple freeze thaw cycles each cycle drops the titer approximately 2 4 fold Limit the number of freeze thaws Sub optimal selection procedure during titration perform an antibiotic kill curve on titration targets prior to titration www clontech com Clontech Laboratories Inc Retroviral Gene Transfer and Expression User Manual X Troubleshooting Guide continued D Infection of Target Cells Poor infection efficiency Target cell viability poor during infection Low expression level Clontech Laboratories Inc Low titer see above section Infection protocol not optimized seeAppendix Cfor references for optimizing transduction protocols Target cells not dividing plate cells at lower con fluency activate with mitogen or use another method to induce cell division Optimize culture conditions for targets prior to infection Packaging ce
34. fficiency gene transfer into rhesus macaque primary T lymphocytes by combining 32 degrees C centrifugation and CH 296 coated plates effect of gene transfer protocol on T cell homing receptor expression Hum GeneTher 12 15 1843 1855 Kotani H Newton P B 3rd Zhang S Chiang Y L Otto E Weaver L Blaese R M Anderson W F amp McGarrity G J 1994 Improved methods of retroviral vector transduction and production for gene therapy Hum Gene Ther 5 1 19 28 Higashikawa F amp Chang L 2001 Kinetic analyses of stability of simple and complex retroviral vectors Virology 280 1 124 131 Centrifugation during transduction Spinoculation believed to counteract diffusion of virus when binding target cells Bunnell B A Muul L M Donahue R E Blaese R M amp Morgan R A 1995 High efficiency retroviral mediated gene transfer into human and nonhuman primate pe ripheral blood lymphocytes Proc Natl Acad Sci USA 92 17 7739 7743 Ohkubo T Barcena A Smith C A Harrison M R amp Muench M O 2001 High efficiency retroviral transduction of fetal liver CD38 CD34 cells implications for in utero and ex utero gene therapy Fetal Diagn Ther 16 5 299 307 Movassagh M Boyer O Burland M C Leclercq V Klatzmann D amp Lemoine F M 2000 Retrovirus mediated gene transfer intoT cells 9596 transduction efficiency without further in vitro selection Hum GeneTher 11 8 1189 1
35. he vector due to toxicity caused by the fusogenic properties of the VSV G protein A positive control cell line GP2 293 Luc allows verification that pVSV G is functioning properly and that target cells can be infected This system takes advantage ofthe envelopes ability to infect non mammalian cells Table l The Retro X Universal Expression System Cat No 631530 also features the GP2 293 cell line To produce infectious virus cotransfect GP2 293 with a retroviral expression vector and the vector that encodes Clontech Laboratories Inc www clontech com Protocol No PT3132 1 Version No PR842519 Retroviral Gene Transfer and Expression User Manual Il Introduction continued the envelope of your choice pAmpho pEco p10A1 or pVSV G A posi tive control vector pOCLIN allows verification that the envelope vector is functioning properly and that target cells can be infected The major advantages of the Universal Packaging System are as follows eThe system can be used with any MMLV based vector eThe tropism can be changed to accomodate the desired cell type e High titers are generated 109 10 e Infectious virus can be obtained in 48 hours and the packaging sys tem is therefore ideal for testing multiple constructs Excess Gag and Pol proteins are not generated which may be detri mental Yap et al 2000 Retroviral Expression Vectors Clontech offers a wide range of retroviral expression vectors that can
36. in human T cells by lentiviral mediated delivery of small interfering RNA against CCR5 Proc Natl Acad Sci USA 100 1 183 8 Quinn T P amp Trevor K T 1997 Rapid quantitation of recombinant retrovirus produced by packaging cell clones Biotechniques 23 1038 1044 Sambrook J amp Russell D W 2001 Molecular Cloning A Laboratory Manual Cold Spring Harbor Laboratory Press Cold Spring Harbor NY Tafuro S Zentilin L Falaschi A amp Giacca M 1996 Rapid retrovirus titration using competi tive polymerase chain reaction Gene Ther 3 679 684 Clontech Laboratories Inc www clontech com Protocol No PT3132 1 Version No PR842519 Retroviral Gene Transfer and Expression User Manual XI References continued Tasaki K Yoshida Y Tagawa M Takenaga K Asano T Ochiai T Isono K Kouzu T Saisho H amp Sakiyama S 1997 Discordant production of released exogenous protein and infec tious virions from retrovirus packaging cells used for gene transduction Anticancer Res 17 4415 4417 Xia H Mao O Paulson H L amp Davidson B L 2002 siRNA mediated gene silencing in vitro and in vivo Nat Biotechnol 20 1006 1010 Yap M W Kingsman S M amp Kingsman A J 2000 Effects of stoichiometry of retroviral components on virus production J Gen Virol 81 2195 2202 Yee J K Friedmann T amp Burns J C 1994 Generation of high titer pseudotyped retroviral vectors wi
37. ines continued C Maintaining Packaging Cell Lines Generally cells should be plated at 10 per 100 mm plate and split every 2 3 days when they reach 70 80 confluency Note Plate HEK 293 based packaging cell lines on collagen coated plates initially to promote adherence after thawing These cells may be cultured on non coated plates flasks after recovery however if adherence is poor we recommend collagen coated vessels for all culturing purposes including viral packaging Split the cells as follows 1 Remove medium and wash cells once with room temperature PBS Note If cells are over confluent omit the wash since the cells may detach from the plate 2 Treat with 2 ml of trypsin EDTA solution for 0 5 1 min Depending on the cell line you may need to treat the cells longer Add 3 ml of media serum to inhibit trypsinization Resuspend cells gently by pipetting 5 Add a predetermined portion of cells to a 100 mm plate in 10 ml of complete medium Rock the plate to distribute the cells evenly Note Split RetroPack PT67 cells at a ratio of up to 1 20 and split HEK 293 based cells at a ratio of 1 10 6 If cell viability is low grow cells for a longer period of time main tain higher cell densities and verify culture conditions D Freezing Packaging Cell Lines Once a stable cell culture is established we recommend that several aliquots of cells be frozen for future use Prepare frozen aliquots of the packagi
38. ium after 24 hr of incubation 6 To determine the efficiency of infection subject a small subpopu lation of cells to antibiotictreatment The infected cells should be used forexperiments orforselectionassoonas possible butnotearlierthan 24 hr after the last infection The growth of some target cells is strongly affected by media con ditioned by the packaging cells You can take certain precautions to avoid an adverse effect induced by the packaging cell derived supernatants e Dilute virus containing media at least 2 fold with fresh medium Expose target cells to the virus for 4 6 hr and then replace with fresh medium o e For cells that prove more difficult to infect please see refer ences located in Appendix C Protocol No PT3132 1 www clontech com Clontech Laboratories Inc Version No PR842519 Retroviral Gene Transfer and Expression User Manual X Troubleshooting Guide A Cloning DNA does not cut as expected Low yield of plasmid Plasmid is difficult to grow or clone B Packaging Cells Poor viability upon thawing Slow growth Cells do not attach to plate Cells appear morphologically different Cannot select for packaging Clontech Laboratories Inc Incomplete digest repeat digest with more enzyme a different enzyme lot or fora longer period oftime Retroviral constructs use a low copy pBR322 ori Grow more liquid culture and purify using low copy purification procedures
39. kaging cells Filter medium through a 0 45 um cellulose acetate or polysulfo nic low protein binding filter Do not use a nitrocellulose filter because nitrocellulose binds proteins in the retroviral membrane and destroys the virus This is the viral stock Prepare six 10 fold serial dilutions as follows a Add 1 35 ml of medium Step 2 to each of six 1 5 ml microcen trifuge tubes b Add 150 ul of virus containing medium Step 4 to the first tube Mix c Transfer 150 ul of viral stock dilution from tube 1 to tube 2 Con tinue serial dilutions by transferring 150 ul of eac hsuccessive dilution to the next prepared tube Infect NIH 3T3 cells by adding 1 ml of the diluted virus medium Step 5 to the wells Final polybrene concentration will be 4 ug ml in 3 ml If you used pLAPSN from the Retro X System for virus produc tion stain cells after 48 hr by assaying for alkaline phosphatase expression Use any standard alkaline phosphatase assay Ausubel et al 1995 For other vectors subject cells to antibiotic selection 24 hr after infection for one week Clontech Laboratories Inc www clontech com Protocol No PT3132 1 Version No PR842519 Retroviral Gene Transfer and Expression User Manual VIII Determining Viral Titer continued See Appendix B for Kill Curve information 8 The viral titer corresponds to the number of colonies present at the highest dilution that contains colonies multiplied by
40. l Curves Appendix C Additional Viral Infection Methods Clontech Laboratories Inc www clontech com Protocol No PT3132 1 2 Version No PR842519 Retroviral Gene Transfer and Expression User Manual Table of Contents continued List of Figures Figure 1 Virus production in packaging cell lines 5 Figure 2 Overview of transient and stable virus production 6 Figure 3 Overview of producing infectious retrovirus 19 List of Tables Table I Host range of packaging cell lines expressing different envelopes 7 Table Il Packaging cell lines to use for infection 8 Table Ill Culture plate conversions 34 Note The viral supernatants produced by these retroviral systems could depending on your DNA insert contain potentially hazardous recombinant virus Due caution must be exercised in the production and handling of recombinant retrovirus The user is strongly advised not to create retroviruses capable of expressing known oncogenes in amphotropic or polytropic host range viruses Please refer to the appropriate regional and institutional guidelines on handling retroviruses Please contact your on site safety officer for specific requirements in your facility In the United States NIH guidelines require that retroviral production and transduction be performed in a Biosafety Level 2 facility For more information see appropriate HHS publications Section IV in this User Manual contains a brief description of Biosafety Level 2 as well as
41. ll line conditioned media may be affecting cell growth dilute viral medium orshorten exposure time to viral supernatant Excessive exposure to polybrene optimize amount of polybrene titrate or shorten exposure time to viral supernatant Low infection efficiency See Section D above Possible promoter inactivation split cells activate with mitogen treat cells with 5 azacytidine Choose a tissue specific promoter Poor cell viability check growth parameters www clontech com Protocol No PT3132 1 Version No PR842519 Retroviral Gene Transfer and Expression User Manual XI References Adeno X Expression System 2000 Clontechniques XV 1 8 10 Ausubel F M Brent R Kingdom R E Moore D M Seidman J G Smith J A amp Struhl K Eds 1995 Current Protocols in Molecular Biology John Wiley amp Sons NY Barton G M amp Medzhitov R 2002 Retroviral delivery of small interfering RNA into primary cells Proc Natl Acad Sci USA 99 23 14943 14945 Brummelkamp T R Bernards R amp Agami R 2002 Stable suppression of tumorigenicity by virus mediated RNA interference Cancer Cell 2 3 243 247 Burns J C Friedmann T Driever W Burrascano M amp Yee J K 1993 Vesicular stomatitis virus G glycoprotein pseudotyped retroviral vectors concentration to very high titer and ef ficient gene transfer into mammalian and nonmammalian cells Proc Natl Acad Sci USA 90 8033 8037
42. minated If less than 10096 of cells carry the construct expression of specified gene may still be detected This efficiency is difficult to achieve in primary cells with transfection e Retroviruses integrate into the hostcell s genome promoting permanent and stable gene transfer as well as persistent expression of the shRNA cassette eViral infection provides consistent reproducible transfer ofthe sequence of interest However transfection efficiency can be low and inconsis tent Clontech Laboratories Inc www clontech com Protocol No PT3132 1 Version No PR842519 Retroviral Gene Transfer and Expression User Manual Il Introduction continued 2 Copy number can be controlled with retroviral vectors The shRNA dosage is important in maintaining gene silencing Barton amp Medzhitov 2002 3 Retroviral vector based shRNAs produced within a retroviral packaging cell do not affect the titer or production of virus particles Brummelkamp et al 2002 4 Because shRNA molecules anneal to specific sequences developing vec torsthattarget specific cell types is unnecessary because only those cells that express the targeted sequence will be affected by the vector 5 Retroviral shRNA expression is more economical than chemical synthesis of small RNA which is expensive for labs to do on a continuous basis 6 Retroviral vector based shRNA expression provides the option for stable expression Adeno X Adenoviral Gene Expre
43. n sites into your gene fragment PCR fragments can be conveniently cloned into any vector using our In Fusion PCR Cloning Kits Digest the vector with the appropriate restriction enzyme s treat with phosphatase and purify Ligate the digested vector and the target gene fragment Transform ligation mixture into E coli Identify the desired recombinant plasmid by restriction analysis and confirm orientation and junctions by sequencing Clontech Laboratories Inc www clontech com Protocol No PT3132 1 Version No PR842519 Retroviral Gene Transfer and Expression User Manual VI Culturing Packaging Cell Lines A General Considerations The protocols in this section are intended for use with packaging cell lines from Clontech e The RetroPack PT67 cell line has a very short doubling time 16 hr Split the culture before it becomes confluent The doubling time for EcoPack2 293 AmphoPack 293 and GP2 293 cell lines is 24 36 hr e If you experience low packaging cell line viability grow the cells for a longer period of time to allow for cell recovery and expansion All our packaging cell lines should be grown at 37 C in a humidified chamber with 5 10 CO See the Certificate of Analysis for details particular to each cell line B Starting Cultures from Frozen Stocks The protocols in this User Manual provide only general guidelines for mammalian cell culture techniques Perform all steps involving c
44. n target cells is not intended to be exclusive Other cells which are not listed may also be infected Virus packaged with the pantropic envelope also infects molusk amphibian ameoba and nematode cells cies or cell type the alternate receptor may still allow viral entry Thus virus packaged by RetroPack PT67 has a broad mammalian host range Table l Miller amp Miller 1994 Miller 1996 These cells are best suited for stable virus production e EcoPack2 293 Cell Line The EcoPack2 293 Packaging Cell Line Cat No 631507 is a human em bryonic kidney HEK 293 derived cell line designed for rapid transient production of high titer ecotropic retrovirus Figure 2 EcoPack2 293 cells can also be used to produce retrovirus stably Bleomycin and hygromycin resistance genes were used to separately introduce the viral gag pol and env genes Virus produced by EcoPack2 293 cells possess an ecotropic envelope gap70 and thus can infect both mouse and rat cells Table I Retroviral sequence within the cell genome has been minimized reducing the likelihood that replication competent virus will be produced through recombination EcoPack2 293 cells are more adherent and produce higher viral titers in comparison to our original EcoPack 293 cells e AmphoPack 293 Cell Line The AmphoPack 293 Packaging Cell Line Cat No 631505 is a human embryonic kidney HEK 293 derived cell line designed for rapid transient Protocol No PT3132
45. ng cells to ensure a renewable source as follows ABO 1 Expand the cell line into the desired number of flasks or plates 2 When the desired number of flasks plates reaches 80 confluency wash the cells once with PBS or HBSS trypsinize using standard tissue culture procedures Freshney 2000 add 2 4 volumes of complete medium to neutralize trypsin and harvest cells 3 Count the cells using a hemocytometer Freshney 2000 and col lect by centrifugation 250 x g for 10 min at room temperature 4 Resuspend in 4 C cell freezing medium containing 10 DMSO at 1 2 x 108 cells ml 5 Dispense 1 ml aliquots into labeled freezing vials and place in a cell freezing container reduces temperature 1 C min at 80 C overnight Alternatively place the vials on ice or at 20 C for 1 2 hr transfer to an insulated container such as a foam ice chest and place in a 80 C freezer for several hours to overnight Transfer vials to liquid nitrogen 7 Two or more weeks later To confirm viability of frozen stocks start a fresh culture of each frozen cell type as described in Section B above Clontech Laboratories Inc www clontech com Protocol No PT3132 1 Version No PR842519 Oo Retroviral Gene Transfer and Expression User Manual VII Virus Production This section provides detailed procedures for virus production target cell infection and stable clone selection For more detailed information or re lated protocols s
46. ntibiotic selection of the infected cells gives rise to a countable number of colonies after roughly 10 14 days Some variations of this method describe a transduction followed by a shorter selection period 3 days Byun et al 1996 recently infected target cells Tafuro et al 1996 Miyao et al 1995 and in situ PCR PRINS Claudio et al 2001 with similar results Other markers include LacZ EGFP Cashion et al 1999 Muldoon et al 1997 and luciferase Although it relates directly to the infectious viral particles functional titer does not provide a consistent measurement of virion concentra tion because it depends upon the transduction efficiency of the cell line being used to determine titer Therefore direct quantitation for determining virus particle concentration may be more desirable Also physical quantitation lends itself for more high throughput applica tions such as screening of stable virus producing clones for high titer variants Direct quantitation of virus concentration in supernatant does not rely on antibiotic selection and therefore all viruses regardless of sequence can be quantitated Methods for the direct quantitation Protocol No PT3132 1 www clontech com Clontech Laboratories Inc Version No PR842519 Retroviral Gene Transfer and Expression User Manual VIII Determining Viral Titer continued IX of virus particles include slot blots Nelson et a 1998 Murdoch et al 1997 Onodera et
47. of six 10 cm tissue culture dishes containing 10 ml of the appropriate complete medium plus varying amounts 0 50 100 200 400 800 ug ml of hygromycin or G418 For puromycin add the drug at 0 1 2 5 5 75 and 10 ug ml b Incubate the cells for 10 14 days replacing the selective medium every four days or more often if necessary c Examine the dishes for viable cells every two days For selecting stable transformants use the lowest concentration that begins to give massive cell death in 5 days and kills all the cells within two weeks 2 Determine optimal plating density Once you have determined the optimal drug concentration deter mine the optimal plating density by plating cells at several different densities in the presence of a constant amount of drug If cells are plated at too high a density they will reach confluency before the selection takes effect Optimal plating density is dependent on population doubling time and cell surface area For example cells that double rapidly have a lower optimal plating density than cells that double slowly a Plate cells at several different densities in each of six 10 cm tissue culture dishes containing 10 ml of the appropriate selective medium Suggested densities cells 10 cm dish 5 x 10 1 x 106 5 x 105 2 x 105 1 x 105 and 5 x 10 b Incubate the cells for 5 14 days replacing the selective medium every four days c Examine the dishes for viable cells every two
48. or expression vectors without the need for subcloning This strategy provides easy access to retroviral expression as well as fluorescent protein tagging yeast two hybrid studies tetracycline regulated gene expression bacterial expression and more Creator Acceptor Vectors such as pLP LNCX and pLP RevTRE serve as entry points into our standard retroviral expression and tetracycline regulated retroviral expression systems See Section XII for ordering information Further details on the Creator System including the Creator DNA Cloning Kits User Manual PT3460 1 are available at www clontech com Retroviral Delivery of RNAi Constructs Our pSIREN Retro Q Vector is designed for gene silencing experiments based on the RNA interference phenomenon July 2003 Clontechniques Expression of silencing RNAs shRNA can decrease expression of a target gene in vivo Xia et al 2002 For more information about gene silencing technology and use of the pSIREN vectors please refer to the Knockout RNAi Systems User Manual PT3739 1 Viral delivery of shR NAs has the following advantages 1 Retroviral expression systems are capable of highly efficient gene delivery Viral vectors take advantage of viral mechanisms that allow efficient delivery of their nucleic acids to susceptible cell targets Since recom binant viruses can infect nearly 10096 of a cell population the selection process used to enrich the population for a construct can be eli
49. ove cellular debris at 500 x g for 10 min Pool all similar stocks at this time 2 Aliquotcleared supernatant into single use tubes to avoid multiple freeze thaw cycles 3 Store tubes at 70 C No cryoprotectant is required Note Avoid multiple freeze thaw cycles since titers can drop as much as 2 4 fold with each cycle Higashikawa amp Chang 2001 Kwon et al 2003 Protocol No PT3132 1 www clontech com Clontech Laboratories Inc Version No PR842519 23 Retroviral Gene Transfer and Expression User Manual VIII Determining Viral Titer A General Considerations Determining the viral titer is necessary for three reasons Confirmation that viral stocks are viable Determinationofthepropertransductionconditionsforyourparticular cell type by adjusting the MOI for the desired transduction efficiency i e control of copy number MOI No of virus particles per target cell Determination of the maximum number of target cells that can be infected for a given virus volume B Procedure for Determining ViralTiter 1 Plate NIH 3T3 cells one day prior to beginning this procedure Plate cells in 6 well plates at a density of 0 5 1 x 10 cells per well Add 2 ml of medium per well Prepare 20 ml of complete medium and add 60 ul of 4 mg ml poly brene Note Polybrene is a polycation that reduces charge repulsion between the virus and the cellular membrane Collect virus containing medium from pac
50. own oncogenes in amphotropic dualtropic or pantropic packaging cell lines Appropriate NIH regional and institutional guidelines apply as well as spe cific guidelines for other countries Please contact your on site safety officer for specific requirements in your facility In the United States NIH guidelines requirethat retroviral production andtransduction be performed in a Biosafety Level 2 BL2 facility A brief description of BL2 is given below It is neither detailed nor complete More information about BL2 guidelinesis available at http bmbl od nih gov contents htm and more information about the risk group assessment for our viral systems is available at http www4 od nih gov oba rac guidelines guidelines html If possible observe and learn the practices described below from someone who has experience working with retroviruses For more information see the following reference e Biosafety in Microbiological and Biomedical Laboratories Fourth Edition May 1999 HHS Pub No CDC 93 8395 U S Department of Health and Human Services PHS CDC NIH Practices e Perform work in a limited access area e Post biohazard warning signs Minimize production of aerosols e Decontaminate potentially infectious wastes before disposal e Take precautions with sharps Safety equipment e Use a laminar flow hood with a HEPA filter e Wear protective laboratory coat face protection and double gloves Facilities e Autoclave for decont
51. roviral vectors with multiple drug selection markers and a complementary helper free packaging cell line Nucleic Acids Res 18 3587 3590 Muldoon R R Levy J P Kain S R Kitts P A amp Link C J Jr 1997 Tracking and quantitation of retroviral mediated transfer using a completely humanized red shifted green flourescent protein gene Biotechniques 22 162 167 Murdoch B Pereira D S Wu X Dick J E amp Ellis J 1997 A rapid screening procedure for the identification of high titer retrovirus packaging clones Gene Ther 4 744 749 Nelson D M Wahlfors J J Chen L Onodera M amp Morgan R A 1998 Characterization of diverse viral vector preparations using a simple and rapid whole virion dot blot method Hum Gene Ther 9 2401 2405 Onodera M Yachie A Nelson D M Welchlin H Morgan R A amp Blaese R M 1997 A simple and reliable method for screening retroviral producer clones without selectable mark ers Hum GeneTher 8 1189 1194 Parente M K amp Wolfe J H 1996 Production of increased titer retrovirus vectors from stable producer cell lines by superinfection and concentration Gene Ther 3 756 760 Pear W S Nolan G P Scott M L amp Baltimore D 1993 Production of high titer helper free retroviruses by transient transfection Proc Natl Acad Sci USA 90 18 8392 8396 Qin X E An D S Chen I S amp Baltimore D 2003 Inhibiting HIV 1 infection
52. ssion For experiments requiring transient gene expression in non dividing or difficult to transfect cells we recommend our Adeno X Expression Sys tems These adenovirus based systems enable high level protein expression in a wide variety of cell types dividing or non dividing without the need for plaque purification January 2000 amp April 2003 Clontechniques Protocol No PT3132 1 www clontech com Clontech Laboratories Inc Version No PR842519 11 Retroviral Gene Transfer and Expression User Manual Il List of Components Store cell lines in liquid nitrogen 196 C Store all plasmids and primers at 20 C Retro X System Cat No 631508 e 1 ml RetroPack PT67 Cell Line 2 x 108cells ml 40 ul pLNCX2 Retroviral Vector 0 5 ug ul 40 ul pLXSN Retroviral Vector 0 5 ug ul 40 ul pLAPSN Retroviral Vector 0 5 ug ul e 100 ul pLNCX Seq PCR Primer 20 uM e 100 ul pLXSN Seq PCR Primer 20 uM Retro X Q Vector Set Cat No 631516 20 ug pOCXIN Retroviral Vector 500 ng ul 20 ug pOCXIH Retroviral Vector 500 ng ul 20 ug pOCXIP Retroviral Vector 500 ng ul 20 ug pOCLIN Retroviral Vector 500 ng ul 100 ul 5 pOC Seq PCR Primer 20 uM 100 ul 3 pOC Seq PCR Primer 20 uM LRCX Retroviral Vector Set Cat No 631511 20 ug pLNCX2 Retroviral Vector 0 5 ug ul 20 ug pLHCX Retroviral Vector 0 5 pg ul 20 ug pLPCX Retroviral Vector 0 5 ug ul e 100 ul 5 pLNCX Seq PC
53. th very broad host range Methods Cell Biol 43 99 112 Protocol No PT3132 1 www clontech com Clontech Laboratories Inc Version No PR842519 33 Retroviral Gene Transfer and Expression User Manual Appendix A Culture Plate Conversions TABLE Ill CULTURE PLATE CONVERSION Size of Plate Growth Area Relataive area Recommended cm Volume 96 well 0 04 X 200 yl 24 well 0 25 X 500 ul swe oa nx om amm so mox oom omm n zex som Flasks 25 3X 5 0 ml 75 9X 12 0 ml Relative area is expressed as a factor of the growth area of a 35 mm culture plate Clontech Laboratories Inc www clontech com Protocol No PT3132 1 34 Version No PR842519 Retroviral Gene Transfer and Expression User Manual Appendix B Titration of Antibiotic Stocks Kill Curves Priorto using G418 hygromycin or puromycin to establish stable packaging cell lines it is important to titrate your selection agent stocks to determine the optimal concentration for selection with the chosen cell line This is also important because of lot to lot variation in the potency of these drugs Therefore you should titrate each new lot of antibiotic to determine the optimal concentration We recommend that you perform two experiments for each drug 1 a titration to determine the optimal drug concentration and 2 an experiment to determine the optimal plating density 1 Titrate at fixed cell density a Plate 2 x 10 cells in each
54. the dilu tion factor For example the presence of four colonies in the 109 dilution would represent a viral titer of 4 x 106 4 colony forming units cfu x 10 4 x 108 cfu ml For virus produced from RetroPack PT67 EcoPack2 293 AmphoPack 293 and GP2 293 cells a good viral titer is 2109 cfu ml C Alternative Methods We recommend that you determine viral titer by infecting NIH 3T3 cells with serially diluted viral supernatants produced with a control vector such as pLAPSN part of our Retro X System Cat No 631508 Infect both NIH 3T3 cells and your target cells See Section IX for instructions on infecting NIH 3T3 cells Infecting your target cell line will give you a rough but rapid estimation of infection success You can use your cells of choice to determine viral titer e g HeLa or Mink cells but NIH 3T3 cells are widely accepted as the standard target cell for titer ing retrovirus because of the efficiency at which these cells become infected The same virus preparation can give different apparent titers on different cells lines due to differential receptor expression and cell cycle rates For more information on determining viral titer please refer to Ausubel et al 1995 The method described inthis manual is astandard gene transducing unit assay that measures the functional titer of a particular virus stock the virus ability to infect is assayed Another method is a drug resistance colony assay in which a
55. titer Once clones are isolated withdraw antibiotic from the medium C Concentrating Virus VSV G enveloped virions only Burns et al 1994 1 Remove cell debris and aggregated virus by low speed centrifuga tion for 5 min at 4 C 2 Pellet the virus at 50 000 x g for 90 min at 4 C Remove the super natant 3 Resuspend the virus to 0 5 1 of the original volume inTNE See Additional Materials required and incubate overnight at 4 C Note If desired perform a second round of ultracentrifugation Steps 1 2 4 Determine the viral titers of pre and post concentrated viral supernatants 5 Infect target cells Section IX B 3 D Producing Virus from Stable Packaging Cell Clone PT67 1 Remove clone from liquid nitrogen and follow thaw procedures outlined in Section VI B 2 Culture the clone until cell culture reaches the desired culture volume 3 Retaining one plate for the continuation of the culture plate the remaining cells at 60 8096 confluency in the desired number of culture vessels 4 Viral supernatants can then be harvested in 24 hr intervals until cells are no longer viable Discard all cells once the virus has been harvested Clontech Laboratories Inc www clontech com Protocol No PT3132 1 Version No PR842519 Retroviral Gene Transfer and Expression User Manual VII Virus Production continued E Storage of Viral Stocks 1 Onceviral supernatanthas been collected briefly centrifuge sample to rem
56. tors provide the packaging signal transcription and processing elements and a target gene Inserts of up to 6 5 kb can be efficiently packaged Transfection of the retroviral vector into a packaging cell line produces high titer replication incompetent virus Theviral env gene expressed by the packaging cell line encodesthe envelope protein which determines the range of infectivity tropism of the packaged virus Viral envelopes are classified according to the receptors used to enter host cells For example ecotropic virus can recognize a receptor found on only mouse and rat cells Amphotropic virus recognizes a receptor found on a broad range of mammalian cell types Dualtropic virus recognizes two different receptors found on a broad range of mammalian cell types A pantropic packaging cell line provided a major advancement in retroviral genetransfer as this cell line produces virus that can infect both mammalian and non mammalian cells Burns et al 1993 Using this cell line virions are pseudo typed with the envelope glycoprotein from the vesicular stomatitis virus VSV G Unlike other viral envelope proteins VSV G mediates viral entry through lipid binding and plasma membrane fusion Emi et al 1991 Stable expression of the VSV G envelope protein is toxic thus the packag ing cell line only contains the viral gag and pol genes Virus is produced by transiently cotransfecting a retroviral expression vector and pVSV G into a
57. um GeneTher 10 17 2799 2810 Chuck A S amp Palsson B O 1996 Consistent and high rates of gene transfer can be obtained using flow through transduction over a wide range of retroviral titers Hum Gene Ther 7 6 743 750 G Addition of fibronectin adhesion domains within fibronectin allow binding to both target cells and virions to facilitate co localization Zhou P Lee J Moore P amp Brasky K M 2001 High efficiency gene transfer into rhesus macaque primary T lymphocytes by combining 32 degrees C centrifugation and CH 296 coated plates effect of gene transfer protocol on T cell homing receptor expression Hum GeneTher 12 15 1843 1855 Moritz T Dutt P Xiao X Carstanjen D Vik T Hanenberg H amp Williams D A 1996 Fibronectin improves transduction of reconstituting hematopoietic stem cells by retroviral vectors evidence of direct viral binding to chymotryptic carboxy terminal fragments Blood 88 3 855 862 Hanenberg H Xiao X L Dilloo D Hashino K Kato l amp Williams D A 1996 Co localization of retrovirus and target cells on specific fibronectin fragments increases genetic transduction of mammalian cells Nat Med 2 8 876 882 Protocol No PT3132 1 www clontech com Clontech Laboratories Inc Version No PR842519 37 Retroviral Gene Transfer and Expression User Manual Appendix C Additional Viral Infection Methods continued Bajaj B Lei P amp Andreadis S T 20
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