title: Efficient production of pronuclear embryos in breeding and nonbreeding season for generating transgenic sheep overexpressing TLR4.
abstract: BACKGROUND
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Key Laboratory of Animal Genetics and Breeding of the Ministry of Agriculture, Beijing Key Laboratory for Animal Genetic Improvement, College of Animal Science and Technology, China Agricultural University, Beijing, 100193 China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Li
authors.affiliation: Department of Public Health, Benedictine University, Lisle, IL 60532 USA.
authors.forename: Di
authors.initials: D
authors.lastname: Lian
authors.affiliation: State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, 100101 China.
authors.forename: Shoulong
authors.initials: S
authors.lastname: Deng
authors.affiliation: Tianjin Institute of Animal Sciences, Tianjin, 300381 China.
authors.forename: Xiaosheng
authors.initials: X
authors.lastname: Zhang
authors.affiliation: Tianjin Institute of Animal Sciences, Tianjin, 300381 China.
authors.forename: Jinlong
authors.initials: J
authors.lastname: Zhang
authors.affiliation: Key Laboratory of Animal Genetics and Breeding of the Ministry of Agriculture, Beijing Key Laboratory for Animal Genetic Improvement, College of Animal Science and Technology, China Agricultural University, Beijing, 100193 China.
authors.forename: Wenting
authors.initials: W
authors.lastname: Li
authors.affiliation: Key Laboratory of Animal Genetics and Breeding of the Ministry of Agriculture, Beijing Key Laboratory for Animal Genetic Improvement, College of Animal Science and Technology, China Agricultural University, Beijing, 100193 China.
authors.forename: Hai
authors.initials: H
authors.lastname: Bai
authors.affiliation: Key Laboratory of Animal Genetics and Breeding of the Ministry of Agriculture, Beijing Key Laboratory for Animal Genetic Improvement, College of Animal Science and Technology, China Agricultural University, Beijing, 100193 China.
authors.forename: Zhixian
authors.initials: Z
authors.lastname: Wang
authors.affiliation: Key Laboratory of Animal Genetics and Breeding of the Ministry of Agriculture, Beijing Key Laboratory for Animal Genetic Improvement, College of Animal Science and Technology, China Agricultural University, Beijing, 100193 China.
authors.forename: Hongping
authors.initials: H
authors.lastname: Wu
authors.affiliation: Key Laboratory of Animal Genetics and Breeding of the Ministry of Agriculture, Beijing Key Laboratory for Animal Genetic Improvement, College of Animal Science and Technology, China Agricultural University, Beijing, 100193 China.
authors.forename: Juncai
authors.initials: J
authors.lastname: Fu
authors.affiliation: Key Laboratory of Animal Genetics and Breeding of the Ministry of Agriculture, Beijing Key Laboratory for Animal Genetic Improvement, College of Animal Science and Technology, China Agricultural University, Beijing, 100193 China.
authors.forename: Hongbing
authors.initials: H
authors.lastname: Han
authors.affiliation: Tianjin Institute of Animal Sciences, Tianjin, 300381 China.
authors.forename: Jianzhong
authors.initials: J
authors.lastname: Feng
authors.affiliation: Key Laboratory of Animal Genetics and Breeding of the Ministry of Agriculture, Beijing Key Laboratory for Animal Genetic Improvement, College of Animal Science and Technology, China Agricultural University, Beijing, 100193 China.
authors.forename: Guoshi
authors.initials: G
authors.lastname: Liu
authors.affiliation: Key Laboratory of Animal Genetics and Breeding of the Ministry of Agriculture, Beijing Key Laboratory for Animal Genetic Improvement, College of Animal Science and Technology, China Agricultural University, Beijing, 100193 China.
authors.forename: Ling
authors.initials: L
authors.lastname: Lian
authors.affiliation: Key Laboratory of Animal Genetics and Breeding of the Ministry of Agriculture, Beijing Key Laboratory for Animal Genetic Improvement, College of Animal Science and Technology, China Agricultural University, Beijing, 100193 China.
authors.forename: Zhengxing
authors.identifier: 0000-0002-2060-0324
authors.initials: Z
authors.lastname: Lian
keywords: Growth; Microinjection; Pronuclear embryos; Sheep; Superovulation