Efficient production of pronuclear embryos in breeding and nonbreeding season for generating transgenic sheep overexpressing TLR4

Yan Li, Di Lian, Shoulong Deng, Xiaosheng Zhang, Jinlong Zhang, Wenting Li, Hai Bai, Zhixian Wang, Hongping Wu, Juncai Fu, Hongbing Han, Jianzhong Feng, Guoshi Liu, Ling Lian, Zhengxing Lian

Brucella is a zoonotic Gram-negative pathogen that causes abortion and infertility in ruminants and humans. TLR4 is the receptor for LPS which can recognize Brucella and initiate antigen-presenting cell activities that affect both innate and adaptive immunity. Consequently, transgenic sheep over-expressing TLR4 are an suitable model to investigate the effects of TLR4 on preventing Brucellosis. In this study, we generated transgenic sheep overexpressing TLR4 and aimed to evaluate the effects of different seasons (breeding and non-breeding season) on superovulation and the imported exogenous gene on growth. In total of 43 donor ewes and 166 recipient ewes in breeding season, 37 donor ewes and 144 recipient ewes in non-breeding season were selected for super-ovulation and injected embryo transfer to generate transgenic sheep. Our results indicated the no. of embryos recovered of donors and the rate of pronuclear embryos did not show any significant difference between breeding and non-breeding seasons (P > 0.05). The positive rate of exogenous TLR4 tested were 21.21 % and 22.58 % in breeding and non-breeding season by Southern blot. The expression level of TLR4 in the transgenic sheep was 1.5 times higher than in the non-transgenic group (P < 0.05). The lambs overexpressing TLR4 had similar growth performance with non-transgenic lambs, and the blood physiological parameters of transgenic and non-transgenic were both in the normal range and did not show any difference. Here we establish an efficient platform for the production of transgenic sheep by the microinjection of pronuclear embryos during the whole year. The over-expression of TLR4 had no adverse effect on the growth of the sheep.