CHO cell endogenous temperature-sensitive promoter and application thereof
A temperature-sensitive, promoter technology, applied in applications, animal cells, reproductive tract cells, etc., can solve the problems of cell stagnation, promoter cell cycle dependence, low activity, etc.
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Embodiment 1
[0092] Example 1 Construction of pLUT-off-X-EGFP recombinant lentiviral vector
[0093] Build process such as figure 1 Shown:
[0094] Using pLEGFP-C1 (Clontech) as a template, the EcoRI-EGFP-XhoI fragment was amplified by Seq PF and Seq PR primers (see Table 1 for the sequence), and the PCR product and pLUT-off vector were double digested with EcoRI and XhoI to obtain EGFP fragment with cohesive ends and pLUT-off linearized vector. Use T4 ligase to ligate the fragments and the linearized vector to obtain the ligation product. For the ligation method, refer to the product instructions. Then use the heat shock method to transform the ligation product into E. coli TOP10 competent cells, screen the positive monoclonals by two steps of bacterial liquid PCR and enzyme digestion identification, and carry out DNA sequencing verification on the screened positive monoclonals to obtain pLUT with the correct sequence -off-EGFP recombinant vector.
[0095] Using the CHO-K1 cell genome...
Embodiment 2
[0099] The construction of embodiment 2 lentiviral cell bank (cell pool)
[0100] Lentiviral packaging:
[0101] (1) One day before co-transfection, 0.3×10 5 HEK 293T cells per mL and 10mL DMEM complete medium (containing 4mM / L glutamine, 10% FBS, the same below) were inoculated in 25T cell culture flasks, and cultured in a carbon dioxide incubator; It was found that the cells were in good condition, and when the confluence was about 80%, they could be used for transfection experiments.
[0102] (2) Add 4.6 μg core plasmid, 2.7 μg psPAX2 vector plasmid and 2.7 μg pMD2.G vector plasmid (mass ratio is about 5:3:3) into serum-free DMEM medium, mix gently; then add 20 μL transfection medium Stain Reagent TurboFect TM Transfection Reagent (Invitrogen, Cat#R0531) was gently mixed to make a final volume of 1 mL; it was allowed to stand at room temperature for 20 minutes to finally form a DNA-transfection reagent complex. Among them, the above-mentioned core plasmid is pLUT-off-X-...
Embodiment 3
[0123] Example 3 Low Temperature Induction Experiment of Virus Infected Cell Bank
[0124] Cell recovery culture: CD FortiCHO TM Meidum (Gibco, Cat#A1148301) complete medium (containing 8mM L-glutamine, the same below) resuscitated containing pLUT-off-X-EGFP vector (wherein X is respectively CIRP-P867, CIRP-P2588, PDI-P598, S100a6-dS18) and pLEGFP-C1 vector (Clontech, Cat#6058-1) cell bank; wherein, the pLEGFP-C1 vector is a recombinant vector driven by a CMV strong promoter to express the EGFP reporter gene. The construction process of the cell bank containing the pLEGFP-C1 vector is as in Example 2, except that the packaging plasmids are gag-pol (Addgene) and pMD2.G.
[0125] Low temperature induction experiment: put the freshly recovered cell bank at 37°C, 110rpm, 8% CO 2 After 2 generations of normal culture in the environment, the cells returned to the normal growth state. Cells in normal growth state were divided into 1×10 6 Cells / mL were inoculated into 5 mL of CDF...
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