HDAC8 gene knockout BHK-21 cell line as well as construction method and application thereof
A BHK-21, gene knockout technology, applied in the field of genetic engineering, can solve the problem of unclear effect, and achieve the effect of accelerating the replication rate and increasing the virus titer
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[0034] According to the HDAC8 gene sequence of golden hamster (Mesocricetus auratus) in NCBI, the gRNA sequence was designed in the first exon region of HDAC8 using CRISPOR software (http: / / crispor.tefor.net / ): GCTCTTCTGATCGGCCCGGG. The designed gRNA was synthesized, annealed and ligated to the PX459 (Addgene#62988) plasmid according to the published method of Zhang Feng's laboratory (Nature Protocols, 2013). The correctly sequenced CRISPR plasmids were extracted and used. According to the standard transfection procedure of Invitrogen Lipofectamine 2000, CRISPR plasmid was transfected into BHK-21 cell line. After 48 hours of transfection, puromycin with a final concentration of 3 μg / mL was added for 5-7 days, and the cells were counted and plated with 100 cells and 300 cells After a week, single cell clones were formed and single clones were picked with a cloning ring, transferred to 24-well plates, and expanded for culture. Different cell clones were collected for identifica...
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