Generation of transgenic cattle by lentiviral gene transfer into oocytes
Generation of transgenic cattle by lentiviral gene transfer into oocytes
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DOI：
10.1095/biolreprod.104.028472
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发表时间：
2004-08-01
影响因子：
3.6
通讯作者：
Pfeifer, A
中科院分区：
文献类型：
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作者：
Hofmann, A;Zakhartchenko, V;Pfeifer, A
转基因牛的潜在益处包括从生产大量的药物相关蛋白质到农业改良。然而，由于传统的DNA显微注射技术效率低下，目前转基因牛的生产是耗时和昂贵的。在这里，我们报告了使用慢病毒高效产生转基因牛。最初尝试通过植入前胚胎的慢病毒感染来生产转基因牛并不成功。相反，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，然后体外受精，导致转基因小牛的出生。此外，通过卵母细胞感染产生的所有小牛都是转基因的，并且通过体内成像和Western印迹检测，这些动物中100%表达eGFP。此外，通过感染胎儿成纤维细胞，然后将细胞核转移到去核卵母细胞中来产生转基因小牛。总之，在调整慢病毒转基因牛后，实现了前所未有的高转基因和表达率。
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.