pCMV-VSV-G

pCMV-VSV-G

pCMV-VSV-G逆病毒载体基本信息：

pCMV-VSV-G载体质粒图谱和多克隆位点信息：

pCMV-VSV-G载体简介：

双质粒系统逆病毒包装质粒pCMV-VSV-G使用方法

Day 1

1.      Plate 5 x 105 293T cells in 6 cm2 dishes containing 5 mL of media. (This can be scaled up if desired)

Day 2

1.      Set up (use polypropylene tubes for this; polystyrene tubes DO NOT work!):

           1 µg retroviral DNA encoding gene X

           1 µg packaging plasmid Mix

                the packaging plasmid (pUMVC3) at a 8:1 ratio with the envelope plasmid (pCVM-VSV-G)-a total of 1µg

           DME without serum to 94μL total

           6 μL Fugene

       Mix and wait 15 to 30 minutes at room temperature

       Add to 293T cells without touching the sides of the dish (DO NOT CHANGEMEDIA)

       If you are using amphotropic virus then move immediately to BL2+ in a secondary container, which has an absorbent material.

       (This does not mean a couple of hours; it means Immediately!).  The rest of this protocol is the same for all viruses---the BL2+ safety practices are in place if you are using amphotropic viruses

Day 3

1.      Change the media to whatever media you wish to use when infecting target cells. 293T cells are easily detached so remember not to put the media directly onto to cells, but rather “run” it down the side of the dish. Remember that you will get the highest titer virus when your cells are “happy.”

2.      Plate out your target cells

Day 4

1.      Remove the medium from the 293T cells and use a 0.45 u syringe filter to remove any 293T cells. DO NOT use the 0.2 u filter, as it is likely to shear the envelope from your virusmaking it noninfectious

        Note: After filtering, the filter should be removed and placed in the biohazard bag in the hood and the syringe rinsed with bleach and decontaminated for a minimum of 20 min. It is useful to place a plastic beaker with bleach in the hood in advance

2.      Add 8 to 10 μg/ml of polybrene (Hexadimethrine bromide) or protamine sulfate to the target cells

3.      Carry out infection for 1 to 4 hours. Remove virus and replace with fresh media

        Note: If you wish to do a second infection the following day, it is important to put fresh media on the cells and not let the virus remain on the cells overnight. The media contains huge amounts of envelope, both associated and unassociated with viral particles, which will bind all the cell surface receptors required for virus adsorption, resulting in their down-regulation. Hence, if you don’t change the media after the initial infection, very few receptors will be available for the next round of infection. In addition, very few cells tolerate the presence of high levels of the VSV-G envelope for extended periods of time (i.e. a lot of your cells may die)

Day 5 +

1.      Allow the cells to recover and begin to express the virus-encoded genes. The cells usually require 48 hours for this to occur

2.      Add drug if you are scoring for the presence of a vector that carries a drug resistance marker. Prior to this step it is advisable that you titrate the drug to be used for selection in order to know precisely how much to add. In addition, it is necessary to bring an extra plate of uninfected cells (often referred to as “canaries”) which will function as a positive control in the kill assay.  Add drug to both plates. When your canaries are dead, you can remove the drug.

pCMV-VSV-G载体序列：

ORIGIN

    1 GAGCTTGGCC CATTGCATAC GTTGTATCCA TATCATAATA TGTACATTTA TATTGGCTCA

   61 TGTCCAACAT TACCGCCATG TTGACATTGA TTATTGACTA GTTATTAATA GTAATCAATT

  121 ACGGGGTCAT TAGTTCATAG CCCATATATG GAGTTCCGCG TTACATAACT TACGGTAAAT

  181 GGCCCGCCTG GCTGACCGCC CAACGACCCC CGCCCATTGA CGTCAATAAT GACGTATGTT

  241 CCCATAGTAA CGCCAATAGG GACTTTCCAT TGACGTCAAT GGGTGGAGTA TTTACGGTAA

  301 ACTGCCCACT TGGCAGTACA TCAAGTGTAT CATATGCCAA GTACGCCCCC TATTGACGTC

  361 AATGACGGTA AATGGCCCGC CTGGCATTAT GCCCAGTACA TGACCTTATG GGACTTTCCT

  421 ACTTGGCAGT ACATCTACGT ATTAGTCATC GCTATTACCA TGGTGATGCG GTTTTGGCAG

  481 TACATCAATG GGCGTGGATA GCGGTTTGAC TCACGGGGAT TTCCAAGTCT CCACCCCATT

  541 GACGTCAATG GGAGTTTGTT TTGGCACCAA AATCAACGGG ACTTTCCAAA ATGTCGTAAC

  601 AACTCCGCCC CATTGACGCA AATGGGCGGT AGGCGTGTAC GGTGGGAGGT CTATATAAGC

  661 AGAGCTCGTT TAGTGAACCG TCAGATCGCC TGGAGACGCC ATCCACGCTG TTTTGACCTC

  721 CATAGAAGAC ACCGGGACCG ATCCAGCCTC CGGTCGACCG ATCCTGAGAA CTTCAGGGTG

  781 AGTTTGGGGA CCCTTGATTG TTCTTTCTTT TTCGCTATTG TAAAATTCAT GTTATATGGA

  841 GGGGGCAAAG TTTTCAGGGT GTTGTTTAGA ATGGGAAGAT GTCCCTTGTA TCACCATGGA

  901 CCCTCATGAT AATTTTGTTT CTTTCACTTT CTACTCTGTT GACAACCATT GTCTCCTCTT

  961 ATTTTCTTTT CATTTTCTGT AACTTTTTCG TTAAACTTTA GCTTGCATTT GTAACGAATT

 1021 TTTAAATTCA CTTTTGTTTA TTTGTCAGAT TGTAAGTACT TTCTCTAATC ACTTTTTTTT

 1081 CAAGGCAATC AGGGTATATT ATATTGTACT TCAGCACAGT TTTAGAGAAC AATTGTTATA

 1141 ATTAAATGAT AAGGTAGAAT ATTTCTGCAT ATAAATTCTG GCTGGCGTGG AAATATTCTT

 1201 ATTGGTAGAA ACAACTACAC CCTGGTCATC ATCCTGCCTT TCTCTTTATG GTTACAATGA

 1261 TATACACTGT TTGAGATGAG GATAAAATAC TCTGAGTCCA AACCGGGCCC CTCTGCTAAC

 1321 CATGTTCATG CCTTCTTCTC TTTCCTACAG CTCCTGGGCA ACGTGCTGGT TGTTGTGCTG

 1381 TCTCATCATT TTGGCAAAGA ATTCCTCGAC GGATCCCTCG AGGAATTCTG ACACTATGAA

 1441 GTGCCTTTTG TACTTAGCCT TTTTATTCAT TGGGGTGAAT TGCAAGTTCA CCATAGTTTT

 1501 TCCACACAAC CAAAAAGGAA ACTGGAAAAA TGTTCCTTCT AATTACCATT ATTGCCCGTC

 1561 AAGCTCAGAT TTAAATTGGC ATAATGACTT AATAGGCACA GCCTTACAAG TCAAAATGCC

 1621 CAAGAGTCAC AAGGCTATTC AAGCAGACGG TTGGATGTGT CATGCTTCCA AATGGGTCAC

 1681 TACTTGTGAT TTCCGCTGGT ATGGACCGAA GTATATAACA CATTCCATCC GATCCTTCAC

 1741 TCCATCTGTA GAACAATGCA AGGAAAGCAT TGAACAAACG AAACAAGGAA CTTGGCTGAA

 1801 TCCAGGCTTC CCTCCTCAAA GTTGTGGATA TGCAACTGTG ACGGATGCCG AAGCAGTGAT

 1861 TGTCCAGGTG ACTCCTCACC ATGTGCTGGT TGATGAATAC ACAGGAGAAT GGGTTGATTC

 1921 ACAGTTCATC AACGGAAAAT GCAGCAATTA CATATGCCCC ACTGTCCATA ACTCTACAAC

 1981 CTGGCATTCT GACTATAAGG TCAAAGGGCT ATGTGATTCT AACCTCATTT CCATGGACAT

 2041 CACCTTCTTC TCAGAGGACG GAGAGCTATC ATCCCTGGGA AAGGAGGGCA CAGGGTTCAG

 2101 AAGTAACTAC TTTGCTTATG AAACTGGAGG CAAGGCCTGC AAAATGCAAT ACTGCAAGCA

 2161 TTGGGGAGTC AGACTCCCAT CAGGTGTCTG GTTCGAGATG GCTGATAAGG ATCTCTTTGC

 2221 TGCAGCCAGA TTCCCTGAAT GCCCAGAAGG GTCAAGTATC TCTGCTCCAT CTCAGACCTC

 2281 AGTGGATGTA AGTCTAATTC AGGACGTTGA GAGGATCTTG GATTATTCCC TCTGCCAAGA

 2341 AACCTGGAGC AAAATCAGAG CGGGTCTTCC AATCTCTCCA GTGGATCTCA GCTATCTTGC

 2401 TCCTAAAAAC CCAGGAACCG GTCCTGCTTT CACCATAATC AATGGTACCC TAAAATACTT

 2461 TGAGACCAGA TACATCAGAG TCGATATTGC TGCTCCAATC CTCTCAAGAA TGGTCGGAAT

 2521 GATCAGTGGA ACTACCACAG AAAGGGAACT GTGGGATGAC TGGGCACCAT ATGAAGACGT

 2581 GGAAATTGGA CCCAATGGAG TTCTGAGGAC CAGTTCAGGA TATAAGTTTC CTTTATACAT

 2641 GATTGGACAT GGTATGTTGG ACTCCGATCT TCATCTTAGC TCAAAGGCTC AGGTGTTCGA

 2701 ACATCCTCAC ATTCAAGACG CTGCTTCGCA ACTTCCTGAT GATGAGAGTT TATTTTTTGG

 2761 TGATACTGGG CTATCCAAAA ATCCAATCGA GCTTGTAGAA GGTTGGTTCA GTAGTTGGAA

 2821 AAGCTCTATT GCCTCTTTTT TCTTTATCAT AGGGTTAATC ATTGGACTAT TCTTGGTTCT

 2881 CCGAGTTGGT ATCCATCTTT GCATTAAATT AAAGCACACC AAGAAAAGAC AGATTTATAC

 2941 AGACATAGAG ATGAACCGAC TTGGAAAGTA ACTCAAATCC TGCACAACAG ATTCTTCATG

 3001 TTTGGACCAA ATCAACTTGT GATACCATGC TCAAAGAGGC CTCAATTATA TTTGAGTTTT

 3061 TAATTTTTAT GAAAAAAAAA AAAAAAAACG GAATTCCTCG AGGGATCCGT CGAGGAATTC

 3121 ACTCCTCAGG TGCAGGCTGC CTATCAGAAG GTGGTGGCTG GTGTGGCCAA TGCCCTGGCT

 3181 CACAAATACC ACTGAGATCT TTTTCCCTCT GCCAAAAATT ATGGGGACAT CATGAAGCCC

 3241 CTTGAGCATC TGACTTCTGG CTAATAAAGG AAATTTATTT TCATTGCAAT AGTGTGTTGG

 3301 AATTTTTTGT GTCTCTCACT CGGAAGGACA TATGGGAGGG CAAATCATTT AAAACATCAG

 3361 AATGAGTATT TGGTTTAGAG TTTGGCAACA TATGCCCATA TGCTGGCTGC CATGAACAAA

 3421 GGTTGGCTAT AAAGAGGTCA TCAGTATATG AAACAGCCCC CTGCTGTCCA TTCCTTATTC

 3481 CATAGAAAAG CCTTGACTTG AGGTTAGATT TTTTTTATAT TTTGTTTTGT GTTATTTTTT

 3541 TCTTTAACAT CCCTAAAATT TTCCTTACAT GTTTTACTAG CCAGATTTTT CCTCCTCTCC

 3601 TGACTACTCC CAGTCATAGC TGTCCCTCTT CTCTTATGGA GATCCCTCGA CGGATCGGCC

 3661 GCAATTCGTA ATCATGTCAT AGCTGTTTCC TGTGTGAAAT TGTTATCCGC TCACAATTCC

 3721 ACACAACATA CGAGCCGGAA GCATAAAGTG TAAAGCCTGG GGTGCCTAAT GAGTGAGCTA

 3781 ACTCACATTA ATTGCGTTGC GCTCACTGCC CGCTTTCCAG TCGGGAAACC TGTCGTGCCA

 3841 GCTGCATTAA TGAATCGGCC AACGCGCGGG GAGAGGCGGT TTGCGTATTG GGCGCTCTTC

 3901 CGCTTCCTCG CTCACTGACT CGCTGCGCTC GGTCGTTCGG CTGCGGCGAG CGGTATCAGC

 3961 TCACTCAAAG GCGGTAATAC GGTTATCCAC AGAATCAGGG GATAACGCAG GAAAGAACAT

 4021 GTGAGCAAAA GGCCAGCAAA AGGCCAGGAA CCGTAAAAAG GCCGCGTTGC TGGCGTTTTT

 4081 CCATAGGCTC CGCCCCCCTG ACGAGCATCA CAAAAATCGA CGCTCAAGTC AGAGGTGGCG

 4141 AAACCCGACA GGACTATAAA GATACCAGGC GTTTCCCCCT GGAAGCTCCC TCGTGCGCTC

 4201 TCCTGTTCCG ACCCTGCCGC TTACCGGATA CCTGTCCGCC TTTCTCCCTT CGGGAAGCGT

 4261 GGCGCTTTCT CATAGCTCAC GCTGTAGGTA TCTCAGTTCG GTGTAGGTCG TTCGCTCCAA

 4321 GCTGGGCTGT GTGCACGAAC CCCCCGTTCA GCCCGACCGC TGCGCCTTAT CCGGTAACTA

 4381 TCGTCTTGAG TCCAACCCGG TAAGACACGA CTTATCGCCA CTGGCAGCAG CCACTGGTAA

 4441 CAGGATTAGC AGAGCGAGGT ATGTAGGCGG TGCTACAGAG TTCTTGAAGT GGTGGCCTAA

 4501 CTACGGCTAC ACTAGAAGAA CAGTATTTGG TATCTGCGCT CTGCTGAAGC CAGTTACCTT

 4561 CGGAAAAAGA GTTGGTAGCT CTTGATCCGG CAAACAAACC ACCGCTGGTA GCGGTGGTTT

 4621 TTTTGTTTGC AAGCAGCAGA TTACGCGCAG AAAAAAAGGA TCTCAAGAAG ATCCTTTGAT

 4681 CTTTTCTACG GGGTCTGACG CTCAGTGGAA CGAAAACTCA CGTTAAGGGA TTTTGGTCAT

 4741 GAGATTATCA AAAAGGATCT TCACCTAGAT CCTTTTAAAT TAAAAATGAA GTTTTAAATC

 4801 AATCTAAAGT ATATATGAGT AAACTTGGTC TGACAGTTAC CAATGCTTAA TCAGTGAGGC

 4861 ACCTATCTCA GCGATCTGTC TATTTCGTTC ATCCATAGTT GCCTGACTCC CCGTCGTGTA

 4921 GATAACTACG ATACGGGAGG GCTTACCATC TGGCCCCAGT GCTGCAATGA TACCGCGAGA

 4981 CCCACGCTCA CCGGCTCCAG ATTTATCAGC AATAAACCAG CCAGCCGGAA GGGCCGAGCG

 5041 CAGAAGTGGT CCTGCAACTT TATCCGCCTC CATCCAGTCT ATTAATTGTT GCCGGGAAGC

 5101 TAGAGTAAGT AGTTCGCCAG TTAATAGTTT GCGCAACGTT GTTGCCATTG CTACAGGCAT

 5161 CGTGGTGTCA CGCTCGTCGT TTGGTATGGC TTCATTCAGC TCCGGTTCCC AACGATCAAG

 5221 GCGAGTTACA TGATCCCCCA TGTTGTGCAA AAAAGCGGTT AGCTCCTTCG GTCCTCCGAT

 5281 CGTTGTCAGA AGTAAGTTGG CCGCAGTGTT ATCACTCATG GTTATGGCAG CACTGCATAA

 5341 TTCTCTTACT GTCATGCCAT CCGTAAGATG CTTTTCTGTG ACTGGTGAGT ACTCAACCAA

 5401 GTCATTCTGA GAATAGTGTA TGCGGCGACC GAGTTGCTCT TGCCCGGCGT CAATACGGGA

 5461 TAATACCGCG CCACATAGCA GAACTTTAAA AGTGCTCATC ATTGGAAAAC GTTCTTCGGG

 5521 GCGAAAACTC TCAAGGATCT TACCGCTGTT GAGATCCAGT TCGATGTAAC CCACTCGTGC

 5581 ACCCAACTGA TCTTCAGCAT CTTTTACTTT CACCAGCGTT TCTGGGTGAG CAAAAACAGG

 5641 AAGGCAAAAT GCCGCAAAAA AGGGAATAAG GGCGACACGG AAATGTTGAA TACTCATACT

 5701 CTTCCTTTTT CAATATTATT GAAGCATTTA TCAGGGTTAT TGTCTCATGA GCGGATACAT

 5761 ATTTGAATGT ATTTAGAAAA ATAAACAAAT AGGGGTTCCG CGCACATTTC CCCGAAAAGT

 5821 GCCACCTAAA TTGTAAGCGT TAATATTTTG TTAAAATTCG CGTTAAATTT TTGTTAAATC

 5881 AGCTCATTTT TTAACCAATA GGCCGAAATC GGCAAAATCC CTTATAAATC AAAAGAATAG

 5941 ACCGAGATAG GGTTGAGTGT TGTTCCAGTT TGGAACAAGA GTCCACTATT AAAGAACGTG

 6001 GACTCCAACG TCAAAGGGCG AAAAACCGTC TATCAGGGCG ATGGCCCACT ACGTGAACCA

 6061 TCACCCTAAT CAAGTTTTTT GGGGTCGAGG TGCCGTAAAG CACTAAATCG GAACCCTAAA

 6121 GGGAGCCCCC GATTTAGAGC TTGACGGGGA AAGCCGGCGA ACGTGGCGAG AAAGGAAGGG

 6181 AAGAAAGCGA AAGGAGCGGG CGCTAGGGCG CTGGCAAGTG TAGCGGTCAC GCTGCGCGTA

 6241 ACCACCACAC CCGCCGCGCT TAATGCGCCG CTACAGGGCG CGTCCCATTC GCCATTCAGG

 6301 CTGCGCAACT GTTGGGAAGG GCGATCGGTG CGGGCCTCTT CGCTATTACG CCAGCTGGCG

 6361 AAAGGGGGAT GTGCTGCAAG GCGATTAAGT TGGGTAACGC CAGGGTTTTC CCAGTCACGA

 6421 CGTTGTAAAA CGACGGCCAG TGAGCGCGCG TAATACGACT CACTATAGGG CGAATTGGAG

 6481 CTCCACCGCG GTGGCGGCCG CTCTAGA

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