pREP4 - Thermo Fisher Scientific
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1. Methods of Transfection Positive Control Assay for CAT Protein Once you have verified that your gene is cloned in the correct orientation and contains an initiation ATG and a stop codon you are ready to transfect your cell line of choice We recommend that you include the positive control vector and a mock transfection negative control to evaluate your results Plasmid DNA for transfection into eukaryotic cells must be clean and free from phenol and sodium chloride Contaminants will kill the cells and salt will interfere with lipid complexing decreasing transfection efficiency We recommend isolating plasmid DNA using the PureLink HQ Mini Plasmid Purification Kit or the PureLink HiPure Plasmid Midiprep Kit available from Invitrogen see page 11 for ordering information For established cell lines e g HeLa consult original references or the supplier of your cell line for the optimal method of transfection Precisely follow the protocol for your cell line Pay particular attention to medium requirements when to pass the cells and at what dilution to split the cells Further information is provided in Current Protocols in Molecular Biology Reference section page 14 Methods for transfection include calcium phosphate Chen and Okayama 1987 Wigler et al 1977 lipid mediated Felgner et al 1989 Felgner and Ringold 1989 and electroporation Chu et al 1987 Shigekawa and Dower 1988 For high efficienc2. and facilitates cloning SV40 polyadenylation signal Efficient transcription termination and polyadenylation of mRNA EBV origin of replication oriP and nuclear antigen EBNA 1 High copy Episomal replication in primate and canine cell lines Reisman and Sugden 1986 Yates et al 1985 Ampicillin resistance gene B lactamase Selection of vector in E coli pUC origin High copy number replication and growth in E coli Herpes Simplex Virus thymidine kinase TK promoter Allows for efficient high level expression of the hygromycin resistance gene McKnight 1980 Hygromycin resistance gene Selection of stable transfectants in mammalian cells Gritz and Davies 1983 Palmer et al 1987 Herpes Simplex Virus thymidine kinase TK promoter polyadenylation signal Efficient transcription termination and polyadenylation of mRNA pREP4 CAT Map of pREP4 CAT 10 The figure below summarizes the features of the pREP4 CAT vector The nucleotide sequence for pREP4 CAT is available at www invitrogen com or by contacting Technical Support see page 12 532 Las vAygromycin Comments for pREP4 CAT 10849 nucleotides RSV LTR promoter bases 1 571 Chloramphenicol acetyl transferase CAT gene bases 630 1289 SV40 polyadenylation signal bases 1345 1586 OriP bases 2004 3979 EBNA 1 gene complementary strand bases 4280 6207 Ampicillin resistance g
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4. GAAAAGC GGGGCTTCGG GCTTTTGCAT AGGGAGGGGG GGTAACGATG AGTTAGCAAC GGTGGAAGTA AGGTGGTACG GATTGGACGA ACCACTGAAT pREP forward primer Transcriptional start TTGTACGCGG AAATGTAGTC ATGCCTTACA ATCGTGCCTT TCCGCATTGC TTAGGAGTCC TTATGCAATA AGGAGAGAAA ATTAGGAAGG AGAGATATTG 3 end of RSV LTR TACAATAAAC GCCATTTGAC CATTCACCAC ATTGGTGTGC Nhel Hind tll Nhel Noti Xho I Sfi I I I CTGCTAGCAA GCTTGCTAGC GGCCGCTCGA GGCCGGCAAG EBV reverse primer GATACATTGA TGAGTTTGGA CAAACCACAA CTAGAATGCA GTGAAATTTG TGATGCTATT GCTTTATTTG TAACCATTAT Note that there are two Nhe I sites in the polylinker CCTCAGGATA CTCTTGTAGT AAGCACCGTG CAACAGACGG TATA TATTTAAGTG ACCTCCAAGC BamH I GCCGGATCCA GTGAAAAAAA TAGTAGTTTC CTTGCAACAT CATGCCGATT GTCTGACATG e CCTAGCTCGA Kpn Pvu Il l l TGGGTACCAG GACATGATAA TGCTTTATTT SV40 poly A AAGCTGCAAT AAACAAGTTA Continued on next page Cloning into pREP4 Continued E coli Transformation Preparing a Glycerol Stock Applying Selective Pressure Transform your ligation mixtures into a competent recA endA E coli strain e g TOP10 and select transformants on LB plates containing 50 to 100 ug mL ampicillin Select 10 20 clones and analyze for the presence and orientation of the insert After identifying the correct clone purify the colony and make a glycerol stock for long term storage It is also a good i
5. J Fritsch E F and Maniatis T 1989 Molecular Cloning A Laboratory Manual Second Edition Plainview New York Cold Spring Harbor Laboratory Press Shigekawa 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 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 Yates J L Warren N and Sugden B 1985 Stable Replication of Plasmids Derived from Epstein Barr Virus in Various Mammalian Cells Nature 313 812 815 2009 Life Technologies Corporation All rights reserved For research use only Not intended for any animal or human therapeutic or diagnostic use 14 invitrogen Corporate Headquarters 5791 Van Allen Way Carlsbad CA 92008 T 1 760 603 7200 F 1 760 602 6500 E tech supporteinvitrogen com For country specific contact information visit our web site at www invitrogen com
6. d maintain pREP4 use the supplied 0 5 ug uL stock solution in TE pH 8 0 to transform a recA endA E coli strain and select transformants on LB plates containing 50 to 100 ug mL ampicillin Be sure to prepare a glycerol stock of your plasmid containing E coli strain for long term storage see page 4 If you will be recombining your entry clone with a destination vector for mammalian expression your insert should contain a Kozak consensus sequence with an ATG initiation codon for proper initiation of translation Kozak 1987 Kozak 1991 Kozak 1990 An example of a Kozak consensus sequence is provided below Other sequences are possible but the G or A at position 3 and the G at position 4 shown in bold illustrates the most commonly occurring sequence with strong consensus Replacing one of the two bases at these positions provides moderate consensus while having neither results in weak consensus The ATG initiation codon is shown underlined G A NNATGG Continued on next page Cloning into pREP4 Continued Multiple Cloning Site of pREP4 224 284 344 404 464 524 584 644 704 Below is the multiple cloning site for pREP4 Restriction sites are labeled to indicate the cleavage site The multiple cloning site has been confirmed by sequencing and functional testing The sequence of pREP4 is available at www invitrogen com or from contacting Technical Support see page 12 5 end of RSV LTR GGC
7. dea to keep a DNA stock of your plasmid at 20 C 1 Streak the original colony on an LB plate containing 50 pg mL ampicillin Incubate the plate at 37 C overnight 2 Isolate a single colony and inoculate into 1 2 mL of LB containing 50 pg mL ampicillin 3 Grow the culture to mid log phase ODsoo 0 5 0 7 Mix 0 85 mL of culture with 0 15 mL of sterile glycerol and transfer to a cryovial 5 Store at 80 C Take some if not all of the following precautions to prevent your clone from being overrun by background contaminants e Use carbenicillin instead of ampicillin Carbenicillin is more stable than ampicillin and allows for a longer period of selective pressure thus preserving your clones longer e Increase the antibiotic concentration More antibiotic means that your clones will not be overwhelmed by B lactamase buildup e Decrease growth times Minimize the amount of time that a clone can grow in the media so as to avoid B lactamase buildup e Periodically refresh plate media If you suspect that tubes plates may be beginning to fail spin them down remove the old media and replenish the wells with fresh LB media plus glycerol and antibiotic Streak clones on selective preferably carbenicillin LB agar plates After about 12 hours isolate colonies for downstream usage This will isolate your desired clones from potential background contaminants Transfection Introduction Plasmid Preparation
8. eeks after addition of hygromycin Note Cells will divide once or twice in the presence of lethal doses of hygromycin so the effects of the drug may take several days to become apparent Complete inhibition of cell growth can take 2 3 weeks in selective medium Continued on next page Creating Stable Cell Lines Continued Selecting Stable Integrants Maintaining Stable Transfectants Once the appropriate hygromycin concentration is determined generate a stable cell line with your construct 1 Transfect your mammalian host cell line with your pREP4 construct using the desired protocol Remember to include a plate of untransfected cells as a negative control and the pREP4 CAT plasmid as a positive control 24 hours after transfection wash the cells and add fresh medium to the cells 48 hours after transfection split the cells into fresh medium containing hygromycin at the pre determined concentration required for your cell line Split the cells such that they are no more than 25 confluent Replenish selective medium every 3 4 days until hygromycin resistant foci can be identified Pick and expand colonies in 96 or 48 well plates Note that pREP4 is an episomally maintained plasmid Reisman and Sugden 1986 Yates et al 1985 Transfected cells may lose the pREP4 plasmid if they are not maintained under selection or are continuously cultured for long periods of time over six months To prevent loss of pREP4 fr
9. ene bases 6833 7693 pUC origin bases 7702 8477 TK promoter bases 8845 9007 Hygromycin resistance gene bases 9071 10081 TK polyadenylation signal bases 10096 10364 Accessory Products Additional Products The following additional products may be used with the pREP4 vectors For more information visit www invitrogen com or contact Technical Support see page 12 Item Quantity Cat no One Shot TOP10 Chemically Competent E coli 20 reactions C4040 03 One Shot TOP10 Electrocomp E coli 20 reactions C4040 52 Ampicillin 200 mg 11593 027 Carbenicillin 5g 10177 012 PureLink HQ Mini Plasmid Purification Kit 100 preps K2100 01 PureLink HiPure Plasmid Midiprep Kit 25 preps K2100 04 Lipofectamine 2000 Reagent 1 5 mL 11668 019 FAST CAT Chloramphenicol Acetyltransferase 100 assays F 2900 Assay Kit 11 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 Headq
10. ine pREP4 is an episomal mammalian expression vector that uses the Rous Sarcoma Virus long terminal repeat RSV LTR enhancer promoter for transcription of recombinant genes inserted into the multiple cloning site The Epstein Barr Virus replication origin oriP and nuclear antigen encoded by the EBNA 1 gene is carried by the plasmid to permit extrachromosomal replication in human primate and canine cells pREP4 also carries the hygromycin B resistance gene for stable selection in transfected cells pREP4 CAT is provided as a control for the relative level of expression of recombinant proteins in a cell line of interest pREP4 CAT expresses the chloramphenicol acetyl transferase protein from the RSV LTR enhancer promoter pREP4 CAT contains the hygromycin B resistance gene for selection Use the following outline to clone and express your gene of interest in pREP4 1 Consult the multiple cloning site depicted on page 3 to design a strategy to clone your gene into pREP4 2 Ligate your insert into the pREP4 vector and transform into E coli Select transformants on LB plates containing 50 to 100 ug mL ampicillin 3 Analyze transformants for the presence of your insert by restriction digestion 4 Select a transformant with the correct restriction pattern and use sequencing to confirm that the gene is cloned in the proper orientation 5 Transfect the construct into a mammalian cell line using your own method of choice Gene
11. ingold 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 Kozak M 1987 An Analysis of 5 Noncoding Sequences from 699 Vertebrate Messenger RNAs Nucleic Acids Res 15 8125 8148 Kozak M 1991 An Analysis of Vertebrate mRNA Sequences Intimations of Translational Control J Cell Biology 115 887 903 Kozak M 1990 Downstream Secondary Structure Facilitates Recognition of Initiator Codons by Eukaryotic Ribosomes Proc Natl Acad Sci USA 87 8301 8305 McKnight S L 1980 The Nucleotide Sequence and Transcript Map of the Herpes Simplex Virus Thymidine Kinase Gene Nucleic Acids Res 8 5949 5964 Neumann J R Morency C A and Russian K O 1987 A Novel Rapid Assay for Chloramphenicol Acetyltransferase Gene Expression BioTechniques 5 444 447 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 Reisman D and Sugden B 1986 trans Activation of an Epstein Barr Viral Transcriptional Enhancer by the Epstein Barr Viral Nuclear Antigen 1 Mol Cell Biol 6 3838 3846 Sambrook
12. invitrogen pREP4 Catalog no V004 50 Rev date 27 October 2009 Manual part no 280033 MAN0000611 ii Table of Contents Kit Contents and Storage EEN iv MON 1 Product Overview ne adden seu EIS ar 1 Methods Lassa EGGE 2 Cloninginto PREP 4 usanne ae Rn ananas ee 2 BE e EE 5 Creating Stable Cell EE 6 aoo e alo D AA anderer nnen 8 PREFA Vector eie aea EE E E eeh 8 pr ENE ET ERE ER NE eeen EE TTT OPT 10 Accessory Products eege niende See nara cucatudvenehan heiten Eea aaa aaae DoE e aaea EEE EEE EE 11 Technical e susanne she han cvennsuedea vencnonses daden ongedaan lades 12 Purchaser Notification 2 HR ren Hl Sande 13 References iergert Ze AANEREN 14 iii Kit Contents and Storage Shipping and Storage Contents Note Genotype of TOP10 e pREP4 vectors are shipped on wet ice Upon receipt store vectors at 20 C e Store the stab at room temperature e 20 ug of pREP4 is supplied at 0 5 ug uL in 10 mM Tris HCI 1 mM EDTA pH 8 0 in a total volume of 40 pL e 10 ug pREP4 CAT is supplied at 0 5 ug uL in 10 mM Tris HCl 1 mM EDTA pH 8 0 in a total volume of 20 pL e 1 stab of TOP10 E coli We recommend that you prepare a glycerol master stock page 4 prior to using the TOP10 E coli F mcrA A mrr hsdRMS mcrBC 80lacZAM15 Alac 74 recA1 araD139 A ara leu 7697 galU galK rpsL Str endA1 nupG Introduction Product Overview Description of the System Experimental Outl
13. om transfected cells we recommend that you follow these guidelines when working with the cells Always use early passage cells Grow and freeze multiple vials of transfected cells to ensure that you have an adequate supply of early passage cells Always maintain cells in medium containing 50 pg mL hygromycin Do not maintain cells in continuous culture for longer than 6 months Appendix pREP4 Vector Map of pREP4 The figure below summarizes the features of the pREP4 vector The sequence for pREP4 is available at www invitrogen com or by contacting Technical Support see page 12 Oo gt Oo E S T Comments for pREP4 10349 nucleotides RSV LTR promoter bases 1 571 Multiple cloning site bases 575 632 SV40 polyadenylation signal bases 641 882 OriP bases 1332 3306 EBNA 1 gene complementary strand bases 3607 5532 Ampicillin resistance gene bases 6158 7018 pUC origin bases 7027 7802 TK promoter bases 8524 8686 Hygromycin resistance gene bases 8750 9892 TK polyadenylation signal bases 9893 10054 Continued on next page pREP4 Vector Continued functionally tested Features of pREP4 pREP4 10 349 bp contains the following elements All features have been Feature Benefit Rous Sarcoma Virus long terminal Allows for efficient high level repeat RSV LTR expression of your recombinant enhancer promoter protein Multiple cloning site Allows for insertion of your gene
14. orporation 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 on purchasing a license to this product for purposes other than research contact Licensing Department Life Technologies Corporation 5791 Van Allen Way Carlsbad California 92008 Phone 760 603 7200 Fax 760 602 6500 Email outlicensing invitrogen com 13 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 Molecular Biology New York Greene Publishing Associates and Wiley Interscience Chen C and Okayama H 1987 High Efficiency Transformation of Mammalian Cells by Plasmid DNA Molec 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 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 R
15. rate a stable cell line if desired 6 Test for expression of the recombinant gene by western blot analysis or functional assay Methods Cloning into pREP4 Before Starting General Molecular Biology Techniques E coli Strain Transformation Method Maintaining pREP4 Kozak Sequence for Mammalian Expression A diagram is provided on the next page to help you design a cloning strategy for ligating your gene of interest into pREP4 General considerations for cloning and transformation are listed below For help with DNA ligations E coli transformations restriction enzyme analysis purification of single stranded DNA DNA sequencing and DNA biochemistry refer to Molecular Cloning A Laboratory Manual Sambrook et al 1989 or Current Protocols in Molecular Biology Ausubel et al 1994 Many E coli strains are suitable for the propagation of this vector including TOP10 and DH5a We recommend that you propagate vectors containing inserts in E coli strains that are recombination deficient recA and endonuclease A deficient endA For your convenience TOP10 is available as chemically competent or electrocompetent cells from Invitrogen see page 11 for ordering information You may use any method of your choice for transformation Chemical transformation is the most convenient for most researchers Electroporation is the most efficient and the method of choice for large plasmids To propagate an
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17. ting translocation and promoting mistranslation Hygromycin B phospho transferase detoxifies hygromycin B by phosphorylation e Hygromycin is light sensitive Store the liquid stock solution at 4 C protected from exposure to light e Hygromycin is toxic Do not ingest solutions containing the drug e Wear gloves a laboratory coat and safety glasses or goggles when handling hygromycin and hygromycin containing solutions To successfully generate a stable cell line expressing your gene of interest from PREP4 determine the minimum concentration of hygromycin B required to kill your untransfected host cell line Typically concentrations ranging from 10 to 400 ng mL hygromycin are sufficient to kill most untransfected mammalian cell lines We recommend that you test a range of concentrations see protocol below to ensure that you determine the minimum concentration necessary for your host cell line 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 hygromycin 0 10 25 50 100 200 400 ng mL hygromycin 3 Replenish the selective media every 3 4 days and observe the percentage of surviving cells 4 Count the number of viable cells at regular intervals to determine the appropriate concentration of hygromycin that prevents growth within 2 3 w
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19. y transfection in a broad range of mammalian cells use Lipofectamine 2000 Reagent available from Invitrogen see page 11 For more information on Lipofectamine 2000 and other transfection reagents visit www invitrogen com or contact Technical Support see page 12 pREP4 CAT is provided as a positive control vector for mammalian transfection and expression see page 10 and may be used to optimize transfection conditions for your cell line The gene encoding chloramphenicol acetyl transferase CAT is expressed in mammalian cells under the control of the RSV LTR enhancer promoter A successful transfection will result in CAT expression that can be easily assayed see below If you want to assay for CAT protein using an ELISA assay you may use the FAST CAT Chloramphenicol Acetyltransferase Assay Kit available from Invitrogen see page 11 for ordering information Other commercial kits for CAT assay are also available Creating Stable Cell Lines Hygromycin Resistance Hygromycin B Activity Determining Antibiotic Sensitivity pREP4 contains the hygromycin resistance gene for selection of stable cell lines using hygromycin B Test the sensitivity of your mammalian host cell to hygromycin B as natural resistance varies among cell lines General information and guidelines are provided in this section for your convenience Hygromycin B 527 5 MW is an aminocyclitol that inhibits protein synthesis by disrup
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