pMD2.G慢病毒载体使用说明-修订编选.pdf

pMD2.G 编号编号 载体名称载体名称 北京华越洋北京华越洋 VECT23114 pMD2.G pMD2.G 载体基本信息载体基本信息：： 载体名称载体名称: pMD2.G 质粒类型质粒类型: 哺乳动物载体；慢病毒包装载体；双质粒包装系统；信哺乳动物载体；慢病毒包装载体；双质粒包装系统；信 封载体封载体 高拷贝高拷贝/低拷贝低拷贝: 高拷贝高拷贝 克隆方法克隆方法: 限制性内切酶，多克隆位点限制性内切酶，多克隆位点 启动子启动子: CMV 载体大小载体大小: 5824 BP 5 测序引物及序列测序引物及序列: CMV fwd 5CGCAAATGGGCGGTAGGCGTG 3 3 测序引物及序列测序引物及序列: -- 载体标签载体标签: 无无 载体抗性载体抗性: Ampicillin 筛选标记筛选标记: 无无 克隆菌株克隆菌株: DH5 或或 HB101 宿主细胞（系）宿主细胞（系）: 包装细胞系如包装细胞系如 293T 备注备注: 慢病毒包装载体慢病毒包装载体 pMD2.G 是是 2 质粒包装系统的信封质粒质粒包装系统的信封质粒 与与 psPAX2 一起使用，适用范围、使用方法见下文。

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也可以与第三代包装载体 pRSV-Rev、、pMDLg/pRRE 一同一同 使用，构成使用，构成 3 质粒包装系统质粒包装系统 稳定性稳定性: 瞬表达瞬表达 组成型组成型/诱导型诱导型: 组成型组成型 病毒病毒/非病毒非病毒: 非病毒非病毒 pMD2.G 载体质粒图谱和多克隆位点信息载体质粒图谱和多克隆位点信息：： pMD2.G 载体简介：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 g retroviral DNA encoding gene X 1 g packaging plasmid Mix the packaging plasmid (psPAX2) at a 8:1 ratio with the envelope plasmid (pMD2.G)-a total of 1g DME without serum to 94L total 6 L 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 cell 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 g/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 dont 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. pMD2.G 载体序列载体序列：： 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。