中文摘要：

目的以慢病毒作为载体，制作增强型绿色荧光蛋白（EGFP）转基因小鼠，建立慢病毒介导的转基因动物制备技术平台。方法慢病毒包装采用三质粒系统。3个质粒分别为转基因质粒FUGW、病毒结构蛋白表达质粒psPAX2以及病毒包膜蛋白表达质粒pMD2．G。病毒包装时，用磷酸钙沉淀法将三质粒共转染来源于人胚肾细胞系的293FT细胞，培养48h后，收取含病毒的上清，并通过高速离心浓缩病毒。将含浓缩病毒液作梯度稀释后感染293FT细胞，通过流式细胞计数仪测定病毒滴度。用显微注射法将浓缩的病毒液注射至FVB／N小鼠1-细胞期胚胎透明带下。在荧光显微镜下观察胚胎EGFP的表达。将注射后发育至2-细胞期的胚胎移植至假孕受体母鼠，得F0代小鼠。通过紫外光照射观测EGFP在小鼠活体内的表达水平。结果病毒液浓缩前的滴度≥10^6 TU／ml（transducing unit，TU），经过高速离心对病毒进行浓缩和纯化，其滴度达到10^9 TU／ml以上。将浓缩病毒液注射至小鼠1-细胞期胚胎透明带下，注射后胚胎的2-细胞期卵裂率为81．8％（1189／1453），利用荧光显微镜分别在注射后60、84、132h观察胚胎，均发现有较强荧光。在2-细胞期，每一视野下胚胎阳性率〉90％，说明包装的病毒成功并高效地转染小鼠胚胎。胚胎移植后假孕母鼠妊娠率为42．9％（12／28），首建鼠阳性率为60．8％（73／120），转基因小鼠的总体研制效率（转基因小鼠数／注射胚胎数）为5．0％（73／1453）。将F0代EGFP转基因小鼠分别与野生型小鼠交配，在其F1、F2、F3代小鼠中均获得了EGFP阳性小鼠，阳性率分别为91．4％（32／35）、93．8％（30／32）、93．1％（27／29）。结论通过慢病毒载体感染小鼠1-细胞期胚胎可有效地制备转基因小鼠。我们已初步建立了慢病毒介导的转基因小鼠制备技术体系。

英文摘要：

Objective To establish an efficient and reliable method for transgenic mouse production, we performed lentiviral vector-mediated transgenesis in mice. Methods The leniviral vector FUGW was packaged by co-transfection of 293FT cells with two packaging vectors, psPAX2 and pMD2. G. The two packaging vectors provide structural proteins and envelope protein respectively which are necessary for the proper package of pseudotype virus. The tire of the pseudotype virus was tested by FACS after infecting 293FT cells with the solution containing the virus particles. The virus was concentrated by high speed centrifugation, and concentrated virus solution was injected into perivitelline space of mouse 1-cell eggs. EGFP expression in pre-implantation eggs was observed under fluorescent microscope, and that in mouse body under UV light. Results The titre of the primary virus solution was ≥ 10^6 TU/ml, and that after concentration was ≥10^9 TU/ml. The concentrated virus solution efficiently infected mouse eggs after injected into the perivitelline space. On 60, 84, 132 h after injection, EGFP expression was observed in the injected eggs. At 24 h after injection when the majority of the injected eggs was in 2-cell stage, more than 90% of the eggs was EGFP positive under fluorescent microscope. The injected 2-cell eggs was transferred into the oviduct of pseudo-pregnant female mice, and 42. 9% （12/28） of the transferred mice became pregnant. The pregnant recipient mice gave birth to 120 pups,of which 60.8% （73/120） showed visible EGFP expression under UV light. The overall transgenic mouse production efficiency （ transgenic mouse/injected eggs） was 5.0% （73/1453 ）. The EGFP transgenic founder mice were mated with wild-type mice, and EGFP expression was detected in F1, F2 and F3 offspring, indicating that the EGFP expression can be transmitted through germline cells. Conclusion Lentiviral transgenesis is an efficient and reliable method to produce transgenic mice. We have established the technological system for