Tpl2-deficient MDCK cell strain as well as construction method and application thereof
A cell line, defective technology, applied in animal cells, vertebrate cells, urinary tract/kidney cells, etc., can solve the problem of inability to obtain stable and safe high-producing avian influenza virus cell lines, etc., and achieve supernatant blood coagulation efficiency. Increase in valence, overcome genetic instability or insecurity, increase productivity
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Embodiment 1
[0066] Example 1 Constructing the MDCK cell line that stably knocks out TPL2
[0067] (1) Design of sgRNA and synthesis of oligonucleotide chain: design two pairs of guide RNA (sgRNA) at the first exon of Tpl2 gene, add CCGG to the 5' end of the sense strand template, which can be digested with Bbs I The formed cohesive ends are complementary; AAAC is added to the 5' end of the antisense strand template, which is complementary to the cohesive ends formed after Bbs I digestion.
[0068] Table 1 TPL2-sgRNA oligonucleotide sequence
[0069]
[0070] (2) Construction of pUC19-sgRNA vector:
[0071] The single strand of the synthesized oligonucleotide was first annealed to form a double strand, and then constructed and inserted into the pUC19-sgRNA plasmid vector through the Bbs I restriction site, and the constructed plasmid was sequenced, and the sequencing results were as follows: figure 2 As shown, it proves that the pUC19-sgRNA plasmid vector was constructed successfully...
Embodiment 2
[0081] 1. Comparison of growth kinetics between Tpl2 knockout MDCK cells and parental cells
[0082] Spread the wild-type and Tpl2-deficient MDCK cell lines constructed in Example 1 in a 96-well plate at 2000 cells / well, count 3 wells each time, and set up 2 wells that only add medium as control wells, and then place them in 37 °C, 5% CO 2 cultivated under conditions. Using the MTS kit, with time as the horizontal axis and absorbance value (the actual absorbance of the cells = the absorbance of the cell well - the absorbance of the control blank) as the vertical axis, the growth curve was drawn using GraphPad Prisms software. The proliferation activity of the two kinds of cells was detected by the MTS method, and it can be seen from the drawn growth curve that there was no significant difference in the growth rate between the Tpl2 knockout MDCK cells and the parental cells ( Figure 4 ).
[0083] 2. Study on the quality control of cell matrix Tpl2-deficient MDCK cell line f...
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