pMD2.G

pMD2.G

pMD2G载体基本信息

出品公司:	Ybscience
载体名称:	pMD2.G
质粒类型:	哺乳动物载体；慢病毒包装载体；双质粒包装系统；信封载体
高拷贝/低拷贝:	高拷贝
克隆方法:	限制性内切酶，多克隆位点
启动子:	CMV
载体大小:	5824 BP
5' 测序引物及序列:	CMV fwd 5’CGCAAATGGGCGGTAGGCGTG 3’
3' 测序引物及序列:	--
载体标签:	无
载体抗性:	Ampicillin
筛选标记:	无
克隆菌株:	DH5α或 HB101
宿主细胞（系）:	包装细胞系如293T
备注:	慢病毒包装载体pMD2.G是2质粒包装系统的信封质粒与 psPAX2 一起使用，适用范围、使用方法见下文。 也可以与第三代包装载体pRSV-Rev、pMDLg/pRRE一同使用，构成3质粒包装系统。
稳定性:	瞬表达
组成型/诱导型:	组成型
病毒/非病毒:	非病毒

 

pMD2G载体质粒图谱和多克隆位点信息

 

pMD2G载体简介

 

第二代慢病毒包装质粒pMD2.G使用方法-慢病毒包装与转染方法

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 (psPAX2) at a 8:1 ratio with the envelope plasmid (pMD2.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 CHANGE MEDIA)

       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 virus
making 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.

 

pMD2G载体序列

LOCUS       pMD2.G	5824 bp 	DNA	SYN
DEFINITION  pMD2.G
ACCESSION   
KEYWORDS    
SOURCE      
  ORGANISM  other sequences; artificial sequences; vectors.
COMMENT     This file is created by Vector NTI
            http://www.biofeng.com/
COMMENT     VNTAUTHORNAME|biofeng.com|
FEATURES             Location/Qualifiers
     source          1..5824
                     /organism="pMD2.G"
                     /mol_type="other DNA"
     misc_feature    complement(40..59)
                     /label="SV40pro_F_primer"
     promoter        162..738
                     /label="CMV_immearly_promoter"
     misc_feature    241..528
                     /label="CAG_enhancer"
     misc_feature    695..715
                     /label="CMV_fwd_primer"
     promoter        696..765
                     /label="CMV_promoter"
     promoter        708..827
                     /label="CMV2_promoter"
     misc_feature    739..758
                     /label="pCEP_fwd_primer"
     misc_feature    741..765
                     /label="LNCX_primer"
     misc_feature    1015..1398
                     /label="bGlob_int"
     gene            1450..2973
                     /label="vsv-G"
                     /gene="vsv-G"
     CDS             1450..2985
                     /label="ORF frame 1"
     misc_feature    2950..2982
                     /label="VSVG Tag"
     rep_origin      complement(4055..4674)
                     /label="pBR322_origin"
     gene            complement(4829..5689)
                     /label="Ampicillin"
                     /gene="Ampicillin"
     CDS             complement(4829..5689)
                     /label="ORF frame 1"
     promoter        complement(5731..5759)
                     /label="AmpR_promoter"
ORIGIN
    1 ggatcccctg agggggcccc catgggctag aggatccggc ctcggcctct gcataaataa
   61 aaaaaattag tcagccatga gcttggccca ttgcatacgt tgtatccata tcataatatg
  121 tacatttata ttggctcatg tccaacatta ccgccatgtt gacattgatt attgactagt
  181 tattaatagt aatcaattac ggggtcatta gttcatagcc catatatgga gttccgcgtt
  241 acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg
  301 tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg
  361 gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt
  421 acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg
  481 accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg
  541 gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt
  601 ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac
  661 tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg
  721 tgggaggtct atataagcag agctcgttta gtgaaccgtc agatcgcctg gagacgccat
  781 ccacgctgtt ttgacctcca tagaagacac cgggaccgat ccagcctccc ctcgaagctt
  841 acatgtggta ccgagctcgg atcctgagaa cttcagggtg agtctatggg acccttgatg
  901 ttttctttcc ccttcttttc tatggttaag ttcatgtcat aggaagggga gaagtaacag
  961 ggtacacata ttgaccaaat cagggtaatt ttgcatttgt aattttaaaa aatgctttct
 1021 tcttttaata tacttttttg tttatcttat ttctaatact ttccctaatc tctttctttc
 1081 agggcaataa tgatacaatg tatcatgcct ctttgcacca ttctaaagaa taacagtgat
 1141 aatttctggg ttaaggcaat agcaatattt ctgcatataa atatttctgc atataaattg
 1201 taactgatgt aagaggtttc atattgctaa tagcagctac aatccagcta ccattctgct
 1261 tttattttat ggttgggata aggctggatt attctgagtc caagctaggc ccttttgcta
 1321 atcatgttca tacctcttat cttcctccca cagctcctgg gcaacgtgct ggtctgtgtg
 1381 ctggcccatc actttggcaa agcacgtgag atctgaattc aacagagatc gatctgtttc
 1441 cttgacacta tgaagtgcct tttgtactta gcctttttat tcattggggt gaattgcaag
 1501 ttcaccatag tttttccaca caaccaaaaa ggaaactgga aaaatgttcc ttctaattac
 1561 cattattgcc cgtcaagctc agatttaaat tggcataatg acttaatagg cacagccata
 1621 caagtcaaaa tgcccaagag tcacaaggct attcaagcag acggttggat gtgtcatgct
 1681 tccaaatggg tcactacttg tgatttccgc tggtatggac cgaagtatat aacacagtcc
 1741 atccgatcct tcactccatc tgtagaacaa tgcaaggaaa gcattgaaca aacgaaacaa
 1801 ggaacttggc tgaatccagg cttccctcct caaagttgtg gatatgcaac tgtgacggat
 1861 gccgaagcag tgattgtcca ggtgactcct caccatgtgc tggttgatga atacacagga
 1921 gaatgggttg attcacagtt catcaacgga aaatgcagca attacatatg ccccactgtc
 1981 cataactcta caacctggca ttctgactat aaggtcaaag ggctatgtga ttctaacctc
 2041 atttccatgg acatcacctt cttctcagag gacggagagc tatcatccct gggaaaggag
 2101 ggcacagggt tcagaagtaa ctactttgct tatgaaactg gaggcaaggc ctgcaaaatg
 2161 caatactgca agcattgggg agtcagactc ccatcaggtg tctggttcga gatggctgat
 2221 aaggatctct ttgctgcagc cagattccct gaatgcccag aagggtcaag tatctctgct
 2281 ccatctcaga cctcagtgga tgtaagtcta attcaggacg ttgagaggat cttggattat
 2341 tccctctgcc aagaaacctg gagcaaaatc agagcgggtc ttccaatctc tccagtggat
 2401 ctcagctatc ttgctcctaa aaacccagga accggtcctg ctttcaccat aatcaatggt
 2461 accctaaaat actttgagac cagatacatc agagtcgata ttgctgctcc aatcctctca
 2521 agaatggtcg gaatgatcag tggaactacc acagaaaggg aactgtggga tgactgggca
 2581 ccatatgaag acgtggaaat tggacccaat ggagttctga ggaccagttc aggatataag
 2641 tttcctttat acatgattgg acatggtatg ttggactccg atcttcatct tagctcaaag
 2701 gctcaggtgt tcgaacatcc tcacattcaa gacgctgctt cgcaacttcc tgatgatgag
 2761 agtttatttt ttggtgatac tgggctatcc aaaaatccaa tcgagcttgt agaaggttgg
 2821 ttcagtagtt ggaaaagctc tattgcctct tttttcttta tcatagggtt aatcattgga
 2881 ctattcttgg ttctccgagt tggtatccat ctttgcatta aattaaagca caccaagaaa
 2941 agacagattt atacagacat agagatgaac cgacttggaa agtaactcaa atcctgcaca
 3001 acagattctt catgtttgga ccaaatcaac ttgtgatacc atgctcaaag aggcctcaat
 3061 tatatttgag tttttaattt ttatggaatt caccccacca gtgcaggctg cctatcagaa
 3121 agtggtggct ggtgtggcta atgccctggc ccacaagttt cactaagctc gcttccttgc
 3181 tgtccaattt ctattaaagg ttccttggtt ccctaagtcc aactactaaa ctgggggata
 3241 ttatgaaggg ccttgagcat ctggattctg cctaataaaa aacatttatt ttcattgcaa
 3301 tgatgtattt aaattatttc tgaatatttt actaaaaagg gaatgtggga ggtcagtgca
 3361 tttaaaacat aaagaaatga agagctagtt caaaccttgg gaaaatacac tatatcttaa
 3421 actccatgaa agaaggtgag gctgcaaaca gctaatgcac attggcaaca gccctgatgc
 3481 ctatgcctta ttcatccctc agaaaaggat tcaagtagag gcttgatttg gaggttaaag
 3541 tttggctatg ctgtatttta cattacttat tgttttagct gtcctcatga atgtcttttc
 3601 actacccatt tgcttatcct gcatctctca gccttgactc cactcagttc tcttgcttag
 3661 agataccacc tttcccctga agtgttcctt ccatgtttta cggcgagatg gtttctcctc
 3721 gcctggccac tcagccttag ttgtctctgt tgtcttatag aggtctactt gaagaaggaa
 3781 aaacaggggg catggtttga ctgtcctgtg agcccttctt ccctgcctcc cccactcaca
 3841 gtgacccgga atccctcgac atggcagtct agcactagtg cggccgcaga tctgcttcct
 3901 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
 3961 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
 4021 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
 4081 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
 4141 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
 4201 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
 4261 ctcaatgctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
 4321 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
 4381 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
 4441 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
 4501 acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
 4561 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
 4621 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
 4681 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
 4741 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
 4801 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
 4861 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
 4921 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct
 4981 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
 5041 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
 5101 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
 5161 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
 5221 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
 5281 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
 5341 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
 5401 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
 5461 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
 5521 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
 5581 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
 5641 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
 5701 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
 5761 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg
 5821 acgt