Construction of Human Neuromuscular Disease-Related Gene Site-Specific Mutant Cell Line by Cas9 Mutation System
DOI:
https://doi.org/10.18063/gse.v4i1.1110Keywords:
Cas9 Mutation System, Neuromuscular Disease, Mutant Cell lineAbstract
Objective: to construct human neuromuscular disease-related gene site-specific mutant cell line by Cas9 mutation system. Methods: according to the principle of CRISPR/Cas9 target design, the exon region of CXCR4 gene sequence was found in the National Center for Biotechnology Information (NCBI) of the United States. Two sgRNAs were designed. Lenticrisprv2 was used as the vector to construct the lenticrisprv2-sgrna recombinant plasmid, which was transformed into the sensitive stbl3 strain. The monoclonal sequencing was selected to verify and expand the culture of the plasmid, then it was transferred to 293T cells for packaging to a slow virus. The virus was collected and infected with 4T1 cells. The monoclonal cells were isolated and cultured by puromycin screening and limited dilution method. The genomic DNA of the selected monoclonal cells was extracted and the DNA fragment near the knockout site was amplified by PCR and sequenced. Results: one cell line had 6 deletion mutations, including DYSF mutation site of neuromuscular disease gene and HEK293T cell model knocked out by DYSF mutation site of neuromuscular disease gene. Conclusion: the recombinant plasmid targeting CXCR4 gene was obtained by CRISPR/Cas9 system, and the human neuromuscular disease-related gene site-specific mutant cell line was successfully constructed.
References
Xu J, Xu Y, Zhao J, et al. CRISPR/Cas9 system and its application in monocotyledons. Jiangsu Agricultural Sciences 2017; (18): 32-33.
Han Q, Cai H, Xue W, et al. The establishment and application of CRISPR/Cas9 gene transcription activation technology. Science Technology and Engineering 2016; (20): 18-20.
Liu F, Yang Z, Yang Y, et al. Use CRISPR/Cas9 system to construct green fluorescent transgenic zebrafish. Animal Husbandry and Veterinary Medicine 2017; (6): 48-49.
Hua W, Bi Y, Liu X, et al. CRISPR/Cas9 technology for preparation of porcine myostatin knockout cells. Genomics and Applied Biology 2015; (5): 55-56. doi: CNKI:SUN:GXNB.0.2015-05-012.
Li H, Shi Z. Research progress on new CRISPR/Cas9 gene targeting system. Jiangsu Journal of Agriculture 2013; (4): 12-13. doi: 10.3969/j.issn.1000-4440.2013.04.037.
Li J, Du J. CRISPR/Cas9 technology in zebrafish gene modification. Life Science 2015; (1): 45-46. doi: 10.13376/j.cbls/2015005.
Tang X, Wang H, Long Q, et al. The application of CRISPR/Cas9 system in rice genome editing. Molecular Plant Breeding 2017; (3): 74-75. doi: 10.13271/j.mpb.015.000895.
Wu F, Kang X, Zhuang Y, et al. CRISPR/Cas9 mediated construction of CD34 GFP report in 293AD cells. Genomics and Applied Biology 2016; (3): 472-478. doi: 10.13417/j.gab.035.000472.
Qiao L, Pang J, Dang C, et al. CRISPR/Cas9 genome editing technology and its application in Streptomyces. Biotechnology Bulletin 2018; (5): 34-35. doi: 10.13560/j.cnki.biotech.bull.1985.2017-0877.
Li J, Feng W, Wang Z, et al. CRISPR/Cas9 technology and its application in transgenic animals. Chinese Journal of Biotechnology 2015; (6): 28-29. doi: 10.13523/j.cb.20150616.
Han Y, Li Q. CRISPR/Cas9 genome editing technology applied in the treatment of HIV-1 infection. Inheritance 2016; (1): 62-63. doi: 10.16288/j.yczz.15-284.
Yu H, Wu J, Zhai P, et al. Construction of GATA-1 gene knockout K562 cell line based on CRISPR/Cas9 technology. Chinese Journal of Cell Biology 2016; (5): 64-65.
Wang X, Liu D, Ouyang X, et al. Used CRISPR/Cas9 technology to create OsGPRP family gene mutants. Molecular Plant Breeding 2018; (15): 12-13. doi: CNKI:SUN:FZZW.0.2018-15-020.
Zhang Z, Tian H, Luo C, et al. Used CRISPR/Cas9 technology to construct TPH1 knockout cell line of goat mammary epithelial cells. Journal of Agricultural Biotechnology 2018; (8): 72-73. doi: 10.3969/j.issn.1674-7968.2018.08.016.
Zhu J, Long D. Targeted knockout of CAPNS1 gene in human neuroma SK-N-SH cells based on CRISPR/Cas9 system. Life Science Research 2018; (4): 50-51. doi: CNKI:SUN:SMKY.0.2018-04-003.
Cheng G, Dong W, Wu X, et al. Liposome-mediated SV40T gene transfection of oriental voles fibroblasts. China Veterinary Science 2016; (6): 81-82.
Hao C, Zhu X, Zeng Y, et al. Liposome method for the construction of Guangxi Bama minipig transgenic somatic cells and production of transgenic cloned embryos. Genomics and Applied Biology 2014; (1): 102-103.
Yu N, Yang Z, Lin X, et al. Culture of bovine sertoli cells and fibroblasts in vitro and pEGFP-N3 transfection. Journal of Northeast Agricultural University 2014; (9): 79-80.
