Bevacqua R J, Fernandez-Martin R, Canel N G, Gibbons A, Texeira D, Lange F, Vans Landschoot G, Savy V, Briski O, Hiriart M I, Grueso E, Ivics Z, Taboga O, Kues W A, Ferraris S, Salamone D F
Animal Biotechnology Laboratory, Facultad de Agronomia. INPA-CONICET, Buenos Aires University, Buenos Aires, Argentina.
Experimental Station Bariloche, INTA, Bariloche, Argentina.
PLoS One. 2017 Mar 16;12(3):e0174025. doi: 10.1371/journal.pone.0174025. eCollection 2017.
Transgenic domestic animals represent an alternative to bioreactors for large-scale production of biopharmaceuticals and could also provide more accurate biomedical models than rodents. However, their generation remains inefficient. Recently, DNA transposons allowed improved transgenesis efficiencies in mice and pigs. In this work, Tn5 and Sleeping Beauty (SB) transposon systems were evaluated for transgenesis by simple cytoplasmic injection in livestock zygotes. In the case of Tn5, the transposome complex of transposon nucleic acid and Tn5 protein was injected. In the case of SB, the supercoiled plasmids encoding a transposon and the SB transposase were co-injected. In vitro produced bovine zygotes were used to establish the cytoplasmic injection conditions. The in vitro cultured blastocysts were evaluated for reporter gene expression and genotyped. Subsequently, both transposon systems were injected in seasonally available ovine zygotes, employing transposons carrying the recombinant human factor IX driven by the beta-lactoglobulin promoter. The Tn5 approach did not result in transgenic lambs. In contrast, the Sleeping Beauty injection resulted in 2 lambs (29%) carrying the transgene. Both animals exhibited cellular mosaicism of the transgene. The extraembryonic tissues (placenta or umbilical cord) of three additional animals were also transgenic. These results show that transpositional transgenesis by cytoplasmic injection of SB transposon components can be applied for the production of transgenic lambs of pharmaceutical interest.
转基因家畜是大规模生产生物制药的生物反应器的替代方案，并且比啮齿动物能提供更精确的生物医学模型。然而，它们的产生效率仍然很低。最近，DNA转座子提高了小鼠和猪的转基因效率。在这项研究中，通过简单的细胞质注射将Tn5和睡美人（SB）转座子系统用于家畜受精卵的转基因研究。对于Tn5，注射转座子核酸和Tn5蛋白的转座体复合物。对于SB，共注射编码转座子和SB转座酶的超螺旋质粒。利用体外产生的牛受精卵建立细胞质注射条件。对体外培养的囊胚进行报告基因表达评估和基因分型。随后，将两种转座子系统注射到季节性可得的绵羊受精卵中，使用携带由β-乳球蛋白启动子驱动的重组人因子IX的转座子。Tn5方法未产生转基因羔羊。相比之下，睡美人注射产生了2只携带转基因的羔羊（29%）。两只动物均表现出转基因的细胞嵌合现象。另外三只动物的胚外组织（胎盘或脐带）也是转基因的。这些结果表明，通过细胞质注射SB转座子成分进行转座转基因可用于生产具有药用价值的转基因羔羊。
