Hofmann Andreas, Zakhartchenko Valeri, Weppert Myriam, Sebald Heidi, Wenigerkind Hendrik, Brem Gottfried, Wolf Eckhard, Pfeifer Alexander
Department of Pharmacy, Institute for Pharmacology, Center for Drug Research, Ludwig-Maximilians University, Butenandstrasse 5(C), 81377 Munich, Germany.
Biol Reprod. 2004 Aug;71(2):405-9. doi: 10.1095/biolreprod.104.028472. Epub 2004 Mar 24.
The potential benefits of transgenic cattle range from the production of large quantities of pharmaceutically relevant proteins to agricultural improvement. However, the production of transgenic cattle is presently time-consuming and expensive because of the inefficiency of the classical DNA microinjection technique. Here, we report the use of lentiviruses for the efficient generation of transgenic cattle. Initial attempts to produce transgenic cattle by lentiviral infection of preimplantation embryos were not successful. In contrast, infection of bovine oocytes with lentiviral vectors carrying an enhanced green fluorescent protein (eGFP) expression cassette followed by in vitro fertilization resulted in the birth of transgenic calves. Furthermore, all of the calves generated by infection of oocytes were transgenic, and 100% of these animals expressed eGFP as detected by in vivo imaging and Western blotting. In addition, a transgenic calf was produced by infection of fetal fibroblasts followed by nuclear transfer into enucleated oocytes. Taken together, after adjusting lentiviral transgenesis to cattle, unprecedented high transgenesis and expression rates were achieved.
转基因牛的潜在益处涵盖从大量生产与药物相关的蛋白质到农业改良等多个方面。然而，由于传统DNA显微注射技术效率低下，目前转基因牛的生产既耗时又昂贵。在此，我们报告利用慢病毒高效培育转基因牛的方法。最初尝试通过慢病毒感染植入前胚胎来生产转基因牛未获成功。相比之下，用携带增强型绿色荧光蛋白（eGFP）表达盒的慢病毒载体感染牛卵母细胞，随后进行体外受精，结果诞生了转基因小牛。此外，通过感染卵母细胞产生的所有小牛都是转基因的，并且通过体内成像和蛋白质印迹检测发现，这些动物中有100%表达eGFP。另外，通过感染胎儿成纤维细胞，随后将其细胞核移植到去核卵母细胞中，也培育出了一头转基因小牛。综上所述，在对慢病毒转基因技术进行调整以适用于牛之后，实现了前所未有的高转基因率和表达率。
