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| Construction of HEK293T cell line stably expressing ABEmax gene based on PiggyBac transposition system |
| 1. National Research Institute for Family Planning, Beijing, 100081；2. National Human Genetic Resources Center, Beijing, 102206 |
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Abstract To construct a tool cell line that stably expresses the ABEmax gene based on the knocked in the adenine base editing tool ABEmax gene by HEK293T cells PiggyBac (PB) transposition system. Methods: ABEmax gene and antibiotic screening gene were inserted into PB plasmid by homologous recombination method to construct pPB-ABEmax eukaryotic recombinant expression plasmid. The recombinant plasmids and the helper plasmids were co-transfected into HEK293T cells, the cell lines were screened by puromycin resistance, and the monoclonal cells were sorted by flow cytometry. After expanded culture, PCR was used to identify the insertion level of the ABEmax gene, RT-qPCR to identify the expression level of ABEmax, and sgRNA transfection was used to test the editing efficiency of the tool cells. Results: The pPB-ABEmax recombinant expression plasmid was successfully constructed, the HEK293T cell lines with specific insertion of the ABEmax gene were screened out, and the gene editing ability of the HEK293T-ABEmax was verified. Conclusion: Several cell lines stably expressing the adenine base editing tool ABEmax gene were successfully constructed and obtained by the PiggyBac gene editing technology, and which had the same high editing activity compared with normal HEK293T cells transfected with sgRNA and ABEmax.
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| [1] |
LIU Huicong1,2, CAO Xiaofang1, WAN Zirui1,3, AN Lisha1, JIN Xiaohua1, MA Xu1,2. Research on the targeted knockout of the PCSK9 gene by C-to-G basic group editor[J]. 中国计划生育学杂志, 2024, 32(6): 1231-. |
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