反转录病毒载体转染小鼠骨髓细胞

背景：为方便、准确检测外源细胞在体内的存活情况,需在外源细胞转移标记基因。目的：观察多种细胞因子联合刺激下反转录病毒载体对BALB/C×C57BL F1代小鼠骨髓细胞基因转移效率的影响。方法：以PA317-GCGPXSN细胞制备病毒上清,NIH3T3细胞测定病毒滴度后,取经过干细胞因子、白细胞介素3、白细胞介素6预培养刺激后的BALB/C×C57BL F1代小鼠骨髓细胞,实验组实施基因转移,流式细胞仪及PCR方法测定基因转移效率。阴性对照组不做基因转移。阳性对照组为PA317-GCGPXSN细胞株。结果与结论：①病毒滴度为1.9×108CFU/L。②对照组 BALB/C×C57BL F1代小鼠骨髓细胞测定的荧光强度数值为0.63%,实验组为76.04%,两组相比差异有显著性意义（P〈0.01）。PCR方法扩增到了NeoR基因的特异性片断,结果证实细胞因子预刺激后实施基因转移,可有效地将外源基因转移进入BALB/C×C57BL F1代小鼠骨髓细胞基因组中。

Retroviral vector transfection of mouse bone marrow cells

BACKGROUND： Gene transfer of exogenous cells should be done for convenient and accurate detection of exogenous cell survival in the body. OBJECTIVE： To explore the gene transfer efficiency of bone marrow cells of BALB/C×C57BL F1 mice mediated by retroviral vector and stimulated with cytokines. METHODS： The viral supernatants were harvested with PA317-GCGPXSN and detected with NIH3T3. Then gene transfer of experimental group was made on bone marrow cells from BALB/C×C57BL F1 mice as target cells which were stimulated by stem cell factor, interleukin 3, interleukin 6. The gene transfer efficiency was detected by flow cytometry and PCR. Negative group was not treated with gene transfer. Positive group used PA317-GCGPXSN cells as target cells. RESULTS AND CONCLUSION： ①The viral titers were 1.9×108 CFU/L. ②The gene transfer efficiency of the experiment group was 76.04%, whereas that of the control group was 0.63%, there was obviously statistical difference （P 0.01）. PCR amplification procured the specific NeoR gene fragments. Results verified that this method can transfer exogenous gene into bone marrow cell genome of BALB/C×C57BL F1 mice efficiently.