Establishment of cell-based transposon-mediated transgenesis in cattle

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• 作者：Ana P. Alessio^a ; Alejandro E. Fili^a ; Wiebke Garrels^b ; ¹ ; Diego O. Forcato^a ; Marí ; a F. Olmos Nicotra^a ; Ana C. Liaudat^a ; Romina J. Bevacqua^c ; Virginia Savy^c ; Marí ; a I. Hiriart^c ; Thirumala R. Talluri^b ; ² ; Jesse B. Owens^d ; Zoltá ; n Ivics^e ; Daniel F. Salamone^c ; Stefan Moisyadi^d ; Wilfried A. Kues^b ; Pablo Bosch^a ; ^{pbosch@exa.unrc.edu.ar" class="auth_mail" title="E-mail the corresponding author}
• 关键词：Cattle ; PiggyBac ; Sleeping beauty ; Transgenesis ; Transposon
• 刊名：Theriogenology
• 出版年：2016
• 出版时间：15 April 2016
• 年：2016
• 卷：85
• 期：7
• 页码：1297-1311.e2
• 全文大小：2697 K

文摘

Transposon-mediated transgenesis is a well-established tool for genome modification in small animal models. However, translation of this active transgenic method to large animals warrants further investigations. Here, the piggyBac (PB) and sleeping beauty (SB) transposon systems were assessed for stable gene transfer into the cattle genome. Bovine fibroblasts were transfected either with a helper-independent PB system or a binary SB system. Both transposons were highly active in bovine cells increasing the efficiency of DNA integration up to 88 times over basal nonfacilitated integrations in a colony formation assay. SB transposase catalyzed multiplex transgene integrations in fibroblast cells transfected with the helper vector and two donor vectors carrying different transgenes (fluorophore and neomycin resistance). Stably transfected fibroblasts were used for SCNT and on in vitro embryo culture, morphologically normal blastocysts that expressed the fluorophore were obtained with both transposon systems. The data indicate that transposition is a feasible approach for genetic engineering in the cattle genome.