Key Laboratory of Genetics
Key Laboratory of Genetics
Li W.-R.,Xinjiang University |
Liu C.-X.,Key Laboratory of Genetics |
Zhang X.-M.,Key Laboratory of Genetics |
Chen L.,Key Laboratory of Genetics |
And 7 more authors.
FEBS Journal | Year: 2017
Fibroblast growth factor 5 (FGF5) regulates hair length in humans and a variety of other animals. To investigate whether FGF5 has similar effects in sheep, we used clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated 9 (Cas9) to generate loss-of-function mutations with the FGF5 gene in Chinese Merino sheep. A total of 16 lambs were identified with genetic mutations within the targeting locus: 13 lambs had biallelic modifications and three lambs had monoallelic modifications. Characterization of the modifications revealed that 13 were frameshift mutations that led to premature termination, whereas the other three were in-frame deletions. Thus, CRISPR/Cas9 efficiently generated loss-of-function mutations in the sheep FGF5 gene. We then investigated the effect of loss of FGF5 function on wool traits in 12 lambs and found that wool staple length and stretched length of genetically modified (GM) yearling sheep were significantly longer compared with that of wild-type (WT) control animals. The greasy fleece weight of GM yearling sheep was also significantly greater compared with that of WT sheep. Moreover, the mean fiber diameter in GM sheep showed no significant difference compared with WT sheep, suggesting that the increase in greasy fleece weight was likely attributed to the increase in wool length. The results of this study suggest that CRISPR/Cas9-mediated loss of FGF5 activity could promote wool growth and, consequently, increase wool length and yield. © 2017 Federation of European Biochemical Societies.
Zhang Z.,Xinjiang Institute of Ecology and Geography |
Zhang Z.,University of Chinese Academy of Sciences |
Zhang Z.,Xinjiang Academy of Animal Science |
Sun Y.,Xinjiang Academy of Animal Science |
And 8 more authors.
Asian-Australasian Journal of Animal Sciences | Year: 2017
Objective: The vertebral number is associated with body length and carcass traits, which represents an economically important trait in farm animals. The variation of vertebral number has been observed in a few mammalian species. However, the variation of vertebral number and quantitative trait loci in sheep breeds have not been well addressed. Methods: In our investigation, the information including gender, age, carcass weight, carcass length and the number of thoracic and lumbar vertebrae from 624 China Kazakh sheep was collected. The effect of vertebral number variation on carcass weight and carcass length was estimated by general linear model. Further, the polymorphic sites of Vertnin (VRTN) gene were identified by sequencing, and the association of the genotype and vertebral number variation was analyzed by the one-way analysis of variance model. Results: The variation of thoracolumbar vertebrae number in Kazakh sheep (18 to 20) was smaller than that in Texel sheep (17 to 21). The individuals with 19 thoracolumbar vertebrae (T13L6) were dominant in Kazakh sheep (79.2%). The association study showed that the numbers of thoracolumbar vertebrae were positively correlated with the carcass length and carcass weight, statistically significant with carcass length. To investigate the association of thoracolumbar vertebrae number with VRTN gene, we genotyped the VRTN gene. A total of 9 polymorphic sites were detected and only a single nucleotide polymorphism (SNP) (rs426367238) was suggested to associate with thoracic vertebral number statistically. Conclusion: The variation of thoracolumbar vertebrae number positively associated with the carcass length and carcass weight, especially with the carcass length. VRTN gene polymorphism of the SNP (rs426367238) with significant effect on thoracic vertebral number could be as a candidate marker to further evaluate its role in influence of thoracolumbar vertebral number. © 2017 by Asian-Australasian Journal of Animal Sciences.
Liu C.,Xinjiang Laboratory of Animal Biotechnology |
Liu C.,Key Laboratory of Genetics |
Liu C.,Xinjiang University |
Wang L.,Xinjiang Laboratory of Animal Biotechnology |
And 26 more authors.
PLoS ONE | Year: 2013
Background: Low efficiency of gene transfer and silence of transgene expression are the critical factors hampering the development of transgenic livestock. Recently, transfer of recombinant lentivirus has been demonstrated to be an efficient transgene delivery method in various animals. However, the lentiviral transgenesis and the methylation status of transgene in sheep have not been well addressed. Methodology/Principle Findings: EGFP transgenic sheep were generated by injecting recombinant lentivirus into zygotes. Of the 13 lambs born, 8 carried the EGFP transgene, and its chromosomal integration was identified in all tested tissues. Western blotting showed that GFP was expressed in all transgenic founders and their various tissues. Analysis of CpG methylation status of CMV promoter by bisulfate sequencing unraveled remarkable variation of methylation levels in transgenic sheep. The average methylation levels ranged from 37.6% to 79.1% in the transgenic individuals and 34.7% to 83% in the tested tissues. Correlative analysis of methylation status with GFP expression revealed that the GFP expression level was inversely correlated with methylation density. The similar phenomenon was also observed in tested tissues. Transgene integration determined by Southern blotting presented multiple integrants ranging from 2 to 6 copies in the genome of transgenic sheep. Conclusions/Significance: Injection of lentiviral transgene into zygotes could be a promising efficient gene delivery system to generate transgenic sheep and achieved widespread transgene expression. The promoter of integrants transferred by lentiviral vector was subjected to dramatic alteration of methylation status and the transgene expression level was inversely correlative with promoter methylation density. Our work illustrated for the first time that generation of transgenic sheep by injecting recombinant lentivirus into zygote could be an efficient tool to improve sheep performance by genetic modification. © 2013 Liu et al.
PubMed | Key Laboratory of Genetics and Xinjiang University
Type: Journal Article | Journal: Asian-Australasian journal of animal sciences | Year: 2016
Myostatin (MSTN) can negatively regulate the growth and development of skeletal muscle, and natural mutations can cause double-muscling trait in animals. In order to block the inhibiting effect of MSTN on muscle growth, we transferred zinc-finger nucleases (ZFN) which targeted sheep MSTN gene into cultured fibroblasts. Gene targeted colonies were isolated from transfected fibroblasts by serial dilution culture and screened by sequencing. Two colonies were identified with mono-allele mutation and one colony with bi-allelic deletion. Further, we introduced the MSTN-ZFN mRNA into sheep embryos by microinjection. Thirteen of thirty-seven parthenogenetic embryos were targeted by ZFN, with the efficiency of 35%. Our work established the technical foundation for generation of MSTN gene editing sheep by somatic cloning and microinjection ZFN into embryos.
Tian Y.,Shihezi University |
Tian Y.,Xinjiang Laboratory of Animal Biotechnology |
Li W.,Xinjiang Laboratory of Animal Biotechnology |
Li W.,Key Laboratory of Genetics |
And 26 more authors.
Biochemical and Biophysical Research Communications | Year: 2013
A number of gene therapy applications and basic research would benefit from vectors expressing multiple genes. In this study, we constructed 2A peptide based tricistronic lentiviral vector and generated transgenic lambs by injecting lentivirus carrying the tricistronic vector into perivitelline space of zygotes. Of 7 lambs born, 2 lambs (#6 and #7) carried the transgene. However, no fluorescent proteins were identified in transgenic sheep. To investigate why the transgene was silenced in transgenic sheep, we analyzed the methylation status of transgene. The methylation level of CMV promoter was 76.25% in #6, and 64.7% in #7. In the coding region of three fluorescent protein genes, methylation levels were extremely high, with the average level of 98.3% in #6 and 98.4% in #7 respectively. Furthermore, the ratio of GFP+ cells were increased significantly when the fibroblasts derived from the transgenic sheep were treated with 5-azaC and/ or TSA. Our results showed that 2A peptide based tricistronic construct was subjected to hypermethylation in transgenic sheep. Moreover, the silencing could be relieved by treating with methytransferase inhibitor and/or deacetylase inhibitor. © 2013 Elsevier Inc. All rights reserved.