Establishment of Quaking Knockout Mouse Embryonic Fibroblast Cell Line Using CRISPR/Cas9 Technology

doi:10.13560/j.cnki.biotech.bull.1985.2023-0879

Abstract

Abstract:

【Objective】 CRISPR/Cas9 technology was used to generate a mouse embryonic fibroblast cell line（NIH3T3）with a knockout of the Quaking gene and to investigate its impact on NIH3T3 cell proliferation. 【Method】 Initially, two sgRNAs targeting Quaking exons were designed using an online platform, and two CRISPR/Cas9 recombinant lentiviral plasmids targeting the first and second exons of the Quaking gene were constructed successfully. These constructs with pcDNA3.1-Quaking overexpression plasmids were co-transfected into HEK293T cells, and the knockout efficiency of Quaking protein was assessed through Western blot analysis. Subsequently, the recombinant lentiviral plasmid（LentiCRISPRv2-sgRNA1）with high knockout efficiency was co-transfected with auxiliary packaging plasmids into HEK293T cells for lentivirus packaging. After lentiviral transduction of NIH3T3 cells, positive monoclonal cell lines were selected using puromycin. Finally, we confirmed the knockout effect through Western blot and immunofluorescence staining, demonstrating the absence of Quaking protein in these cells. 【Result】 Sequencing confirmed the occurrence of a targeted gene segment deletion. CCK8 assays revealed that Quaking gene knockout significantly inhibited NIH3T3 cell proliferation. 【Conclusion】 This study represents the first successful utilization of CRISPR/Cas9 technology to establish a Quaking gene knockout cell line in mouse embryonic fibroblast cells（NIH3T3）, providing a valuable in vitro model for exploring the mechanistic role of the Quaking gene in the regulation of mouse physiological functions.

Key words: Quaking, CRISPR/Cas9, mouse embryonic fibroblasts, gene knockout

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.

Figures/Tables 9

Table 1 Sequences information

名称Name	序列Sequence（5'-3'）
sgRNA1	CACCGAAGTGCAGAATTGCCTGACG
sgRNA1	AAACCGTCAGGCAATTCTGCACTTC
sgRNA2	CACCGGATCTTCAACCACCTCGAG
sgRNA2	AAACCTCGAGGTGGTTGAAGATCC
Target-F	TTATTTCCCAAAGTGAC
Target-R	ATGCCCGAATAGGTT

Fig. 1 sgRNA design of Quaking gene in mice

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. 5 Morphology of NIH3T3 cells A: NIH3T3 cell morphology in control group; B: Quaking gene knockout NIH3T3 morphology

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. 7 QKI protein expression detected by immunofluorescence staining

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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