Bmal1 gene mutant cell strain as well as construction method and application thereof
A construction method and cell line technology, applied in the field of genetic engineering, can solve problems such as controversial conclusions
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[0019] A method for constructing a Bmal1 gene mutant cell line. The present invention is explained in detail below, but does not mean that the present invention is limited thereto.
[0020] (1) Design sgRNA primer sequences: find the mBmal1 exon sequence found on the NCBI website, use the sgRNA design website of Zhang Feng's laboratory (http: / / crisprp.mit.edu) to find the sgRNA sequence in the exon, The sgRNA sequence with the highest score and the lowest off-target rate was selected according to the sgRNA sequence score. The final designed and synthesized sgRNA of Bmal1 gene was ACCCGTATTTCCCCGTTCGCTGG.
[0021] (2) Construction of px459-mBmal1 vector: digestion of px459 plasmid. px459 5.5μL (1μg), FastDigestBbs1 1uL, 10xFastDigest Buffer 2μL, ddH 2 O, 11.5 μL. Digestion at 37°C for 30 min. Phosphorylation and annealing of both strands of synthetic complementary sgRNA. Oligol (100 mM) 1 μL, Oligo2 (100 mM) 1 μL, 10xT4Ligation Buffer 1 μL, T4 PNK 0.5 μL, ddH 2 O 6.5 μL. ...
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