摘要
目的构建过表达Hes1基因的逆转录病毒载体,并通过感染相对原始的小鼠Lin-造血细胞检测其感染效率。方法通过RT-PCR法从B6小鼠骨髓细胞中扩增Hes1基因功能区,将扩增产物连接入逆转录病毒载体MSCV-ICN1-IRES-GFP,构建重组逆转录病毒载体MSCV-Hes1-IRES-GFP,瞬时转染293T细胞,设转染空载体的细胞为对照组,流式细胞仪检测转染效率,Realtime PCR检测Hes1基因的表达;将重组逆转录病毒载体MSCV-Hes1-IRES-GFP及空载体与Lipofectamine 2000混合后,转染293T细胞,包装重组逆转录病毒,稳定感染小鼠Lin-细胞,设感染空载体病毒的细胞为对照组,流式细胞仪检测感染效率,Realtime PCR检测Hes1基因的表达。结果测序证实重组逆转录病毒载体构建正确;转染293T细胞后第3天,重组逆转录病毒的转染效率约为95%,Hes1基因表达量约为对照组的5倍;病毒上清感染Lin-细胞后第4、5天,重组逆转录病毒的感染效率约为7%,Hes1基因的表达量约为对照组的6倍。结论已成功构建了过表达Hes1基因的重组逆转录病毒载体,并在小鼠Lin-细胞中稳定表达,为研究白血病环境下Hes1对造血干祖细胞的作用奠定了基础。
Objective To construct a retroviral vector for over-expression of mouse HesI gene, and determine the infection efficiency through infection of relatively primitive mouse Lin- hematopoietic cells. Methods Hesl domain was ampli- fied from the bone marrow ceils of B6 mice by RT-PCR and inserted into retroviral vector MSCV-ICNI-IRES-GFP. The constructed recombinant retroviral vector MSCV-Hesl-IRES-GFP was transfected to 293T cells . The transfection efficacy was determined by flow cytometry, using those of cells transfected with empty vector as control, for the expression level of Hesl was determined by realtime PCR. Recombinant retroviral vector MSCV-Hesl-IRES-GFP and empty vector were mixed with Lipofectmaine 2000 respectively, and transfected to 293T cells for packaging of recombinant retrovirus. Mouse Linhematopoietic cells were stably infected with recombinant retrovirus, of which the transfection efficacy was determined by flow cytometry, using those infected with empty vector virus as control. The expression level of Hesl in GFP＋ cells was determined by realtime PCR. Results DNA sequencing confirmed that the recombinant retroviral vector was constructed correctly. The transfeetion efficacy of recombinant retrovirus in 293T cells on day 3 after tranfection was about 95%, while the expression level of Hesl was about 5 times of that in control group. The infection efficacy of recombinant retrovirus in Lincells on days 4 and 5 after infection with virus supernatant was about 7%, while the expression level of Hesl gene was 6 times of that in control group. Conclusion Recombinant retrovirus vector for over-expression of Hesl gene was successfully constructed and expressed stably in mouse Lincells, which laid a foundation of study on effect of Hes 1 on hemopoietic stem/progenitor cells of patients with leukemia.
出处
《中国生物制品学杂志》
CAS
CSCD
2013年第2期205-208,共4页
Chinese Journal of Biologicals
