Biotechnology Bulletin ›› 2024, Vol. 40 ›› Issue (2): 65-72.doi: 10.13560/j.cnki.biotech.bull.1985.2023-0879
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GAO Deng-ke1,2(), MA Bai-rong1,2, GUO Yi-ying1, LIU Wei1,2, LIU Tian1,2, JIN Ya-ping1,2, JIANG Zhou3(), CHEN Hua-tao1,2()
Received:
2023-09-12
Online:
2024-02-26
Published:
2024-03-13
Contact:
JIANG Zhou, CHEN Hua-tao
E-mail:gdk960101@nwafu.edu.cn;jweiz@126.com;htchen@nwafu.edu.cn
GAO Deng-ke, MA Bai-rong, GUO Yi-ying, LIU Wei, LIU Tian, JIN Ya-ping, JIANG Zhou, CHEN Hua-tao. Establishment of Quaking Knockout Mouse Embryonic Fibroblast Cell Line Using CRISPR/Cas9 Technology[J]. Biotechnology Bulletin, 2024, 40(2): 65-72.
名称Name | 序列Sequence(5'-3') |
---|---|
sgRNA1 | CACCGAAGTGCAGAATTGCCTGACG |
AAACCGTCAGGCAATTCTGCACTTC | |
sgRNA2 | CACCGGATCTTCAACCACCTCGAG |
AAACCTCGAGGTGGTTGAAGATCC | |
Target-F | TTATTTCCCAAAGTGAC |
Target-R | ATGCCCGAATAGGTT |
Table 1 Sequences information
名称Name | 序列Sequence(5'-3') |
---|---|
sgRNA1 | CACCGAAGTGCAGAATTGCCTGACG |
AAACCGTCAGGCAATTCTGCACTTC | |
sgRNA2 | CACCGGATCTTCAACCACCTCGAG |
AAACCTCGAGGTGGTTGAAGATCC | |
Target-F | TTATTTCCCAAAGTGAC |
Target-R | ATGCCCGAATAGGTT |
Fig. 2 Construction and identification of recombinant plasmids A: Electrophoresis of LentiCRISPRv2 empty vector enzyme digestion, M: marker; 1: empty carrier of LentiCRISPRv2 after digestion by Esp3I. B: Sequencing results of recombinant plasmids LentiCRISPRv2-sgRNA1 and LentiCRISPRv2-sgRNA2
Fig. 3 Screening of recombinant plasmids with high QKI protein knockout efficiency via Western blot 1: Blank control cells without transfection; 2: transfected pcDNA3.1-Quaking overexpression vector; 3: co-transfected pcDNA3.1-Quaking and LentiCRISPRv2 empty vectors; 4: co-transfected pcDNA3.1-Quaking and LentiCRISPRv2-sgRNA1; 5: co-transfected pcDNA3.1-Quaking and LentiCRISPRv2-sgRNA2
Fig. 4 Schematic diagram of comparison between sequencing results of Quaking knockout cell lines and wild-type cell lines Wild-type: Quaking wild-type cell line sequence; Quaking KO: Quaking knockout cell line sequence; sgRNAs are marked in black, PAM sequences in red, dashed blue lines indicate the number of bases removed, and numbers in parentheses indicate the number of bases removed
Fig. 6 Detection of QKI protein expression via Western blot 1: NIH3T3 cell samples in control group; 2: NIH3T3 cell samples knocked out by Quaking gene
Fig. 8 Effects of Quaking gene knockout on the proliferation ability of NIH3T3 cells P < 0.001 indicates that the cell proliferation activity was significantly different between the two groups in two-factor ANOVA; and ***P < 0.001 indicates a very significant difference
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