| 出品公司: | Addgene |
|---|---|
| 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。 |
| 产品目录号: | -- |
| 稳定性: | 瞬表达 |
| 组成型/诱导型: | 组成型 |
| 病毒/非病毒: | 非病毒 |
买家导航
pMD2G载体基本信息
pMD2G载体图谱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
//
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