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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

Identification of exogenous TLR4 gene and quantification. a The brief structure of 3S-pTLR4 vector, the sizes of the injected TLR4 fragment was 3172 bp. b Southern blot analysis. The 5118 band was the endogenous TLR4 fragment when the sheep genomic DNA was digest by Hind III. Numbered 1, 2, 3, 5 and 7 represent positive individuals. Numbered 4, 6 and 8 represent negative individuals. 2× and 4× are samples of transgenic vectors, here used as positive controls. c The mRNA expression of TLR4 was quantified using real-time PCR, each individual was repeated three times (n = 3). Tg = Transgenic sheep, N = 5. NTg = Non-transgenic sheep, N = 10. Superscript letter (*) represents statistically significant difference (P < 0.05)
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Fig4: Identification of exogenous TLR4 gene and quantification. a The brief structure of 3S-pTLR4 vector, the sizes of the injected TLR4 fragment was 3172 bp. b Southern blot analysis. The 5118 band was the endogenous TLR4 fragment when the sheep genomic DNA was digest by Hind III. Numbered 1, 2, 3, 5 and 7 represent positive individuals. Numbered 4, 6 and 8 represent negative individuals. 2× and 4× are samples of transgenic vectors, here used as positive controls. c The mRNA expression of TLR4 was quantified using real-time PCR, each individual was repeated three times (n = 3). Tg = Transgenic sheep, N = 5. NTg = Non-transgenic sheep, N = 10. Superscript letter (*) represents statistically significant difference (P < 0.05)

Mentions: As shown in Table 1, in total, 310 recipient sheep were transplanted and 80 lambs were born including six dead fetuses, the survival rate was 92.50 %. Subsequently, 64 sheep out of 74 live sheep (Ten sheep were used for other experiment, therefore they were removed from this study) were identified by Southern blot and 14 sheep were positive, the positive rate was 21.88 %, in which, seven transgenic sheep were produced at non-breeding season. Meanwhile, it’s worth noting that one positive sheep was resulted from the reuse of donors as recipients. By real-time PCR, the expression level of TLR4 in the transgenic sheep was 1.5 times higher than the non-transgenic group (P < 0. 05, Fig. 4c).Table 1


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)

Identification of exogenous TLR4 gene and quantification. a The brief structure of 3S-pTLR4 vector, the sizes of the injected TLR4 fragment was 3172 bp. b Southern blot analysis. The 5118 band was the endogenous TLR4 fragment when the sheep genomic DNA was digest by Hind III. Numbered 1, 2, 3, 5 and 7 represent positive individuals. Numbered 4, 6 and 8 represent negative individuals. 2× and 4× are samples of transgenic vectors, here used as positive controls. c The mRNA expression of TLR4 was quantified using real-time PCR, each individual was repeated three times (n = 3). Tg = Transgenic sheep, N = 5. NTg = Non-transgenic sheep, N = 10. 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

Fig4: Identification of exogenous TLR4 gene and quantification. a The brief structure of 3S-pTLR4 vector, the sizes of the injected TLR4 fragment was 3172 bp. b Southern blot analysis. The 5118 band was the endogenous TLR4 fragment when the sheep genomic DNA was digest by Hind III. Numbered 1, 2, 3, 5 and 7 represent positive individuals. Numbered 4, 6 and 8 represent negative individuals. 2× and 4× are samples of transgenic vectors, here used as positive controls. c The mRNA expression of TLR4 was quantified using real-time PCR, each individual was repeated three times (n = 3). Tg = Transgenic sheep, N = 5. NTg = Non-transgenic sheep, N = 10. Superscript letter (*) represents statistically significant difference (P < 0.05)
Mentions: As shown in Table 1, in total, 310 recipient sheep were transplanted and 80 lambs were born including six dead fetuses, the survival rate was 92.50 %. Subsequently, 64 sheep out of 74 live sheep (Ten sheep were used for other experiment, therefore they were removed from this study) were identified by Southern blot and 14 sheep were positive, the positive rate was 21.88 %, in which, seven transgenic sheep were produced at non-breeding season. Meanwhile, it’s worth noting that one positive sheep was resulted from the reuse of donors as recipients. By real-time PCR, the expression level of TLR4 in the transgenic sheep was 1.5 times higher than the non-transgenic group (P < 0. 05, Fig. 4c).Table 1

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