Subjects: Medicine, Pharmacy >> Preclinical Medicine submitted time 2018-06-15 Cooperative journals: 《南方医科大学学报》
Abstract： Objective To construct a cell model of 4.1R gene knockout in murine macrophage cell line RAW264.7 using CRISPR/ Cas9 technique. Methods Three high-grade small-guide RNAs (sgRNAs) that could specifically identify 4.1R gene were synthesized and inserted into lentiCRISPRv2 plasmid. RAW264.7 cells were infected with sgRNA-Cas9 lentivirus from 293T cells transfected with the recombinant sgRNA-lentiCRISPRv2 plasmid, and the positive cells were screened using puromycin and the monoclonal cells were obtained. The expression of 4.1R protein in the monoclonal cells was measured by Western blotting, and the mutation site was confirmed by sequence analysis. Result A 4.1R gene knockout RAW264.7 cell line was obtained, which showed a 19-bp deletion mutation in the 4.1R gene sequence and obviously enhanced proliferation. Conclusion We successfully constructed a 4.1R gene knockout macrophage cell line using CRISPR/Cas9 technique, which may facilitate further investigation of the function of 4.1R in macrophages.
Subjects: Medicine, Pharmacy >> Preclinical Medicine submitted time 2018-01-25 Cooperative journals: 《南方医科大学学报》
Abstract： Objective To construct a cell model of 4.1Rgene knockout in murine macrophage cell line RAW264.7 using CRISPR/ Cas9 technique. Methods Three high-grade small-guide RNAs (sgRNAs) that could specifically identify 4.1R gene were synthesized and inserted into lentiCRISPRv2 plasmid. RAW264.7 cells were infected with sgRNA-Cas9 lentivirus from 293T cells transfected with the recombinant sgRNA-lentiCRISPRv2 plasmid, and the positive cells were screened using puromycin and the monoclonal cells were obtained. The expression of 4.1R protein in the monoclonal cells was measured by Western blotting, and the mutation site was confirmed by sequence analysis. Result A 4.1R gene knockout RAW264.7 cell line was obtained, which showed a 19-bp deletion mutation in the 4.1R gene sequence and obviously enhanced proliferation. Conclusion We successfully constructed a 4.1Rgene knockout macrophage cell line using CRISPR/Cas9 technique, which may facilitatefurtherinvestigationofthefunctionof4.1Rinmacrophages.
Subjects: Medicine, Pharmacy >> Preclinical Medicine submitted time 2017-12-27 Cooperative journals: 《南方医科大学学报》
Abstract： Objective To detect the expression of protein 4.1 family members in mouse melanoma cell lines and evaluate their effect on cell proliferation. Methods PCR and Western blot were used to detected to the expression of protein 4.1 family members (4.1R, 4.1B, 4.1G, and 4.1N) at the mRNA and protein levels in B16 and B16-F10 cell lines. The expression plasmid vector pEGFP-N1-EPB41L3 carrying 4.1B gene sequence amplified from genomic RNA of mouse embryo fibroblasts was constructed and transiently transfected into mouse melanoma cells. The change in cell proliferation was assessed using MTT assay. Results The mRNA and protein expressions of all the protein 4.1 family members, with the exception of 4.1B, were detected in both B16 and B16-F10 cells. Transfection of cells with the eukaryotic expression vector pEGFP-N1-EPB41L3 markedly inhibited cell proliferation as compared with the non-transfected cells. Conclusion The eukaryotic expression vector carrying EPB41L3 sequence is capable of inhibiting the proliferation of mouse melanoma B16 and B16-F10 cells.
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