Construction and function validation of inducible immortalized gene integration vectors
Journal Title: Chinese Journal of Blood Transfusion - Year 2024, Vol 37, Issue 12
Abstract
[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-including 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 inserted 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 transfection, 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 expression 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 vectors provides a technical foundation for establishing conditionally immortalized primary cell lines, thereby facilitating research on the large-scale in vitro production and expansion of blood cells, such as erythrocytes and platelets.
Authors and Affiliations
Wei YUE, Yue YANG, Baohua QIAN, Yanxin LI, Haihui GU
Analysis and evaluation of hepatitis B test results of blood nucleic acid testing under different screening modes
Objective To evaluate the effectiveness of Roche Cobas s 201 in detecting HBV by analyzing its blood nucleic acid testing (NAT) results. Methods The results were grouped according to the enzyme-linked immunosorbent assay...
Practice of blood donor emergency recruitment by TikTok
Objective To discuss the practice of emergency recruitment of blood donors through TikTok by shooting short videos, and evaluate the recruitment effect. Methods The completed short videos about blood donor emergency recr...
Informationization and development strategy in blood collection and supply institutions in Beijing
[Objective] To investigate and analyze the current status, needs and issues of information system construction in blood collection and supply institutions in Beijing, and to provide strategic suggestions for the further...
Establishment and preliminary verification of a highly sensitive nucleic acid extraction method for OBI detection
Objective To establish and verify a new nucleic acid extraction method for OBI detection with large volume and high sensitivity, and apply it in the quantitative determination of OBI samples with low viral load. Methods...
Establishment of reference intervals for serum sTfR and sTfR/lgSF in apparently healthy adults in Wuhan
Objective To establish reference intervals for serum soluble transferrin receptor (sTfR) and sTfR/log serum ferritin index (sTfR/lgSF) in apparently healthy adults in the Wuhan area, so as to provide reference for clinic...