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

Li Y, Lian D, Deng S, Zhang X, Zhang J, Li W, Bai H, Wang Z, Wu H, Fu J, Han H, Feng J, Liu G, Lian L, Lian Z - J Anim Sci Biotechnol (2016)

Bottom Line: 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 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.The over-expression of TLR4 had no adverse effect on the growth of the sheep.

View Article: PubMed Central - PubMed

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.

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.

No MeSH data available.


Related in: MedlinePlus

The production efficiency of in vivo pronuclear embryos in different seasons. a The rate of pronuclear embryo. b The rate of 2-cell. c The rate of transferable embryo. BS = Breeding Season, N = 43. NBS = Non-breeding Season, N = 37. Superscript letter (*) represents statistically significant difference (P < 0.05)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4940989&req=5

Fig2: The production efficiency of in vivo pronuclear embryos in different seasons. a The rate of pronuclear embryo. b The rate of 2-cell. c The rate of transferable embryo. BS = Breeding Season, N = 43. NBS = Non-breeding Season, N = 37. Superscript letter (*) represents statistically significant difference (P < 0.05)

Mentions: In brief, 512 transferable embryos (512 pronuclear embryos, 0 2-cells) and 3 untransferable embryos (In poor quality such as degeneration) were obtained in the breeding season. In addition, 542 transferable embryos (519 pronuclear embryos, 23 2-cells) and 10 untransferable embryos were obtained in the nonbreeding season. The 2-cells embryos were flushed out from the oviduct in vivo during the superovulation operation. They were resulted from cleavage of pronuclear embryo in vitro. For each sheep, the ratio of pronuclear embryos was calculated as the formula: (The no. of pronuclear embryos)/(Total no. of embryos flushed out of oviduct). The average and standard deviation were calculated. The ratio of 2-cells embryos was calculated as above. The transferable embryos included pronuclear and 2-cells embryos. The ratio of transferable embryos was calculated as above too. As shown in Fig. 2, higher rate of pronuclear embryos was observed in the breeding season but there was no significant difference between them (99.03 ± 3.48 % versus 93.56 ± 11.28 %, P > 0.05, Fig. 2a). The rate of 2-cells embryo was lower in breeding season than that in the nonbreeding season but they showed no significant difference (0.00 ± 0.00 % versus 4.29 ± 9.74 %, P > 0.05, Fig. 2b). Furthermore, there were no significant difference in the rate of transferable embryos between different seasons (99.03 ± 3.48 % versus 97.85 ± 5.32 %, P > 0.05, Fig. 2c).Fig. 2


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

Li Y, Lian D, Deng S, Zhang X, Zhang J, Li W, Bai H, Wang Z, Wu H, Fu J, Han H, Feng J, Liu G, Lian L, Lian Z - J Anim Sci Biotechnol (2016)

The production efficiency of in vivo pronuclear embryos in different seasons. a The rate of pronuclear embryo. b The rate of 2-cell. c The rate of transferable embryo. BS = Breeding Season, N = 43. NBS = Non-breeding Season, N = 37. Superscript letter (*) represents statistically significant difference (P < 0.05)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4940989&req=5

Fig2: The production efficiency of in vivo pronuclear embryos in different seasons. a The rate of pronuclear embryo. b The rate of 2-cell. c The rate of transferable embryo. BS = Breeding Season, N = 43. NBS = Non-breeding Season, N = 37. Superscript letter (*) represents statistically significant difference (P < 0.05)
Mentions: In brief, 512 transferable embryos (512 pronuclear embryos, 0 2-cells) and 3 untransferable embryos (In poor quality such as degeneration) were obtained in the breeding season. In addition, 542 transferable embryos (519 pronuclear embryos, 23 2-cells) and 10 untransferable embryos were obtained in the nonbreeding season. The 2-cells embryos were flushed out from the oviduct in vivo during the superovulation operation. They were resulted from cleavage of pronuclear embryo in vitro. For each sheep, the ratio of pronuclear embryos was calculated as the formula: (The no. of pronuclear embryos)/(Total no. of embryos flushed out of oviduct). The average and standard deviation were calculated. The ratio of 2-cells embryos was calculated as above. The transferable embryos included pronuclear and 2-cells embryos. The ratio of transferable embryos was calculated as above too. As shown in Fig. 2, higher rate of pronuclear embryos was observed in the breeding season but there was no significant difference between them (99.03 ± 3.48 % versus 93.56 ± 11.28 %, P > 0.05, Fig. 2a). The rate of 2-cells embryo was lower in breeding season than that in the nonbreeding season but they showed no significant difference (0.00 ± 0.00 % versus 4.29 ± 9.74 %, P > 0.05, Fig. 2b). Furthermore, there were no significant difference in the rate of transferable embryos between different seasons (99.03 ± 3.48 % versus 97.85 ± 5.32 %, P > 0.05, Fig. 2c).Fig. 2

Bottom Line: 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 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.The over-expression of TLR4 had no adverse effect on the growth of the sheep.

View Article: PubMed Central - PubMed

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.

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.

No MeSH data available.


Related in: MedlinePlus