慢病毒介导的外源基因体外投递系统的建立

目的针对不同哺乳类细胞建立相应的慢病毒体外感染体系，以建立慢病毒介导的外源基因体外投递系统。方法按Invitrogen公司推荐的标准程序进行慢病毒（携带EGFP基因）包装（脂质体介导的瞬时转染）、超速离心浓缩和保存等，随后用病毒上清或浓缩后的病毒感染293FT细胞，24—48h后荧光显微镜下观察是否见绿色荧光以证实慢病毒是否成功生产；将携带EGFP基因的病毒上清或浓缩后的病毒分别加入内含293FF细胞、小鼠ES细胞、小鼠胚胎成纤维细胞（MEFs）或小鼠睾丸生殖细胞的培养板孔内，感染6—12h后，用相应培养基替换感染液，数天后荧光显微镜下观察是否见绿色荧光以证实慢病毒是否成功感染不同哺乳类细胞。结果按标准程序生产的携带EGFP基因慢病毒（病毒上清或浓缩后的病毒）成功高效率感染293FF细胞、MEFs或小鼠睾丸生殖细胞；用浓缩后的病毒（携带EGFP基因）感染小鼠ES细胞，亦可获得EGFP阳性的ES细胞克隆。结论熟练掌握了慢病毒包装、浓缩及鉴定等技术，同时针对不同哺乳类细胞建立了相应的慢病毒介导的外源基因体外传递系统，这些为相关后续研究打下了良好的基础。

Establishment of Lentivirus-Mediated in Vitro Gene Delivery System

Objective To establish the lentivirus-mediated in vitro gene delivery systems for different mammalian ceils. Method According to the standard protocol from Invitrogen, lentiviruses carrying EGFP gene were produced or/and concentrated, followed by confirming that lentiviruses harboring EGFP gene were successfully made through EGFP assay under fluorescent stereo microscope after infecting 293FT cells. Based on the different infecting protocols, lentiviruses harboring EGFP gene were employed to infect the different mammalian cells [ i.e., 293Fr cells, mouse embryonic stem cells （mES cells）, mouse embryonic fibroblasts （MEFs） and mouse germ ceils ], followed by EGFP assay under fluorescent stereo microscope 1-4 days after infection. Result 293FT cells, MEFs and mouse germ cells were successfully and efficiently infected by lentivirus supernatant or concentrated lentivirus produced according to the standard protocol from Invitrogen, whereas EGFP-positive mES cell colonies were observed under fluorescent stereo microscope several days after infection when based on the special infecting procedure, the single mES cell was infected by concentrated lentivirus carrying EGFP gene. Conclusion Lentivirus-mediated in vitro gene delivery systems for different mammalian cells were successfully established in our lab.