Construction and function validation of inducible immortalized gene integration vectors
Objective To construct inducible immortalization gene vectors for transfection into primary cells,enabling the establishment of a conditionally immortalized cell line that support their sustained cultivation and proliferation in vitro. Methods Using gene homologous recombination technology,the coding sequences (CDS) of immortalization genes-inclu-ding human telomerase reverse transcriptase (hTERT),simian virus 40 large T antigen (SV40LT),acute myeloid leukemia fusion genes NUP98-KDM5A (N/K) and CBFA2T3-GLIS2 (C/G),as well as the proto-oncogene KRAS were precisely in-serted into the tetracycline (Tet)-inducible eukaryotic expression lentiviral vector pLV2-TRE3GS-EGFP-MCS-3×FLAG-hPGK-Tet-On-SV40-Neo and the transposon PB-TRE3G-3×FLAG-T2A-Puro-SV40-PA. Lentiviral packaging,cell transfec-tion,mRNA expression analysis,Western blotting for protein detection,green fluorescent protein (GFP) visualization,and cell proliferation assays were conducted to evaluate transfection efficiency and assess the regulatory effects of Tet on gene ex-pression in 293T and MEF cells. Results The Tet-inducible lentiviral vectors pLV2-Tet-SV40LT,pLV2-Tet-N/K,and pLV2-Tet-C/G,along with the transposon vectors PB-Tet-hTERT,PB-Tet-SV40LT,PB-Tet-N/K,PB-Tet-C/G,and PB-Tet-KRAS,were successfully constructed. In 293T cells,the expression levels of all target genes were upregulated after transfection. In MEF cells,the immortalizing functions of SV40LT and N/K were validated. By modulating Tet addition,cell proliferation levels were effectively regulated,leading to the successful establishment of conditionally immortalized pLV2-SV40LT-MEF and pLV2-N/K-MEF cell lines. Conclusion The construction of Tet-inducible immortalizing gene vec-tors provides a technical foundation for establishing conditionally immortalized primary cell lines,thereby facilitating re-search on the large-scale in vitro production and expansion of blood cells,such as erythrocytes and platelets.
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