PAlb-uPA slow virus vector and preparation method and application thereof
A lentiviral vector and vector technology, applied in the field of genetic engineering, can solve the problems that hinder the construction, popularization and use of human chimeric liver mouse models, the difficulty of screening, and the high mortality rate of offspring newborn mice, so as to achieve convenient regulation, The effect of simple screening and simple preparation method
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Embodiment 1
[0033] Embodiment 1, PAlb-uPA lentiviral vector construction
[0034] Lenti-X used in the present invention TMTet-on Advanced Inducible Expression System Kit was purchased from Clontech Company (containing pLVX Tight Puro vector and pLVX-Tet-On vector); pMD19-T Simple vocter vector, PCR reaction reagent, reverse transcription kit, in-Fusion Dry-Down PCR cloning kit and DNA polymerase primeSTAR HS were purchased from TaKaRa Company; RNA extraction kit RNAprep pure Tissue Kit and gel recovery kit were purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.; plasmid extraction kit was purchased from Qiagen Company; Trizol extract, RNase H and FV membrane were purchased from Invitrogen; pLKO.1 cloning vector, polybrene Ploybrene and trypsin were purchased from Sigma; DMEM medium, ethylenediaminetetraacetic acid (EDTA) and Opti-MEM medium Purchased from GIBCO; 293T cells, P-eGFP plasmid and H2.35 mouse hepatocytes (ATCC) were preserved by our Institute.
[0035] 1. JG...
Embodiment 2
[0041] Example 2, PAlb-uPA lentiviral vector functional verification
[0042] 1. PAlb-uPA lentiviral vector virus packaging
[0043] Take well-grown 293T cells, digest and wash them, and use Opti-MEM selection medium to make cell suspension, then transfer them to different culture dishes for further culture, and set them aside; then take two 50mL sterile centrifuge tubes, of which Add 0.5 mL of the PAlb-uPA lentiviral vector JGY-PLV at a concentration of 0.5 mg / mL, 1 mL of the viral envelope protein expression plasmid pMD2.G at a concentration of 0.2 mg / mL and 0.5 mL of the Viral structural protein expression plasmid psPAX2, then add Opti-MEM medium to make up to 18mL, and set aside; add 0.5 mL Fran-EZ solution and 17.5 mL Opti-MEM medium to the other tube, mix well, then add to the first test tube, Shake while adding until it is completely added to obtain plasmid transfection solution. Then, the obtained plasmid transfection solution was added dropwise to the cell culture...
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