Juncheng Huang

Selection of ovine oocytes by brilliant cresyl blue staining.

Sheep oocytes derived from the ovaries collected from the slaughterhouse are often used for research on in vitro embryo production, animal cloning, transgenesis, embryonic stem cells, and other embryo biotechnology aspects. Improving the in vitro culture efficiency of oocytes can provide more materials for similar studies. Generally, determination of oocyte quality is mostly based on the layers of cumulus cells and cytoplasm or cytoplasm uniformity and colors. This requires considerable experience to better identify oocyte quality because of the intense subjectivity involved (Gordon (2003), Madison et al. (1992) and De Loos et al. (1992)). BCB staining is a function of glucose-6-phosphate dehydrogenase (G6PD) activity, an enzyme synthesized in developing oocytes, which decreases in activity with maturation. Therefore, unstained oocytes (BCB−) are high in G6PD activity, while the less mature oocytes stains are deep blue (BCB+) due to insuffcient G6PD activity to decolorize the BCB dye.

Highly Efficient Generation of Transgenic Sheep by Lentivirus Accompanying the Alteration of Methylation Status

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.

The critical role of myostatin in differentiation of sheep myoblasts.

Myostatin [MSTN, also known as growth differentiation factor 8 (GDF8)], is an inhibitor of skeletal muscle growth. Blockade of MSTN function has been reported to result in increased muscle mass in mice. However, its role in myoblast differentiation in farm animals has not been determined. In the present study, we sought to determine the role of MSTN in the differentiation of primary sheep myoblasts. We found that ectopic overexpression of MSTN resulted in lower fusion index in sheep myoblasts, which indicated the repression of myoblast differentiation. This phenotypic change was reversed by shRNA knockdown of the ectopically expressed MSTN in the cells. In contrast, shRNA knockdown of the endogenous MSTN resulted in induction of myogenic differentiation. Additional studies revealed that the induction of differentiation by knocking down the ectopically or endogenously expressed MSTN was accompanied by up-regulation of MyoD and myogenin, and down-regulation of Smad3. Our results demonstrate that MSTN plays critical role in myoblast differentiation in sheep, analogous to that in mice. This study also suggests that shRNA knockdown of MSTN could be a potentially promising approach to improve sheep muscle growth, so as to increase meat productivity.

Derivation and characterization of ovine embryonic stem-like cell lines in semi-defined medium without feeder cells.

Domestic animal embryonic stem (ES) cells would provide an invaluable research tool for genetic breeding and the production of transgenic animals. Unfortunately, authentic domestic animals ES cells have not been established despite progress made over more than two decades. Here, we show that ovine ES-like cells can be efficiently derived and propagated in a semi-defined medium that contains N2, B27, GSK3 inhibitor (CHIR99021), and basic fibroblast growth factor (bFGF). These ovine ES-like cells had a characteristic three-dimensional appearance, showed a bFGF dose-dependence, expressed specific markers such as alkaline phosphatase (AP), Oct-4, Sox2, Nanog and can be maintained for 30 passages. Moreover, these cells differentiated in vitro into neuronal cells, and formed teratomas containing a variety of different tissues including cartilage and neural tissue when injected into kidney capsules of severe combined immunodeficiency (SCID) mice. But the cell lines fail to contribute to embryonic development upon blastocyst transplantation. To our knowledge, this is the first experiment to use semi-defined medium without feeder-cells to derive ES-like cells from ovine blastocysts, and opens the door to deriving authentic ES cells from domesticated ungulates.

Expression of 2A peptide mediated tri-fluorescent protein genes were regulated by epigenetics in transgenic sheep.

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.

Knockdown of endogenous myostatin promotes sheep myoblast proliferation

Myostatin (MSTN), is a known negative regulator of myogenesis. Silencing of the function of MSTN could result in increasing muscle mass in mice. To determine the function of endogenous MSTN expression on proliferation of sheep myoblasts, a short-hairpin RNA-targeting sheep MSTN was constructed into lentiviral vector to silence endogenous MSTN expression. We demonstrated that silencing of endogenous MSTN gene with up to approximately 73.3% reduction by short hairpin RNA (shRNA) resulted in significant increase (overall 28.3%) of proliferation of primary ovine myoblasts. The upregulation of proliferation was accompanied by the decrease expression of MyoD (−37.6%, p = 0.025), myogenin (−33.1%, p = 0.049), p21 (−49.3%, p = 0.046), and Smad3 (−50.0%, p = 0.007). Silencing of myostatin using shRNA may provide a feasible approach to improve meat productivity in farm animals.

Caffeine and dithiothreitol delay ovine oocyte ageing

The intracellular glutathione levels and developmental competence of aged oocytes after parthenogenetic activation, somatic cell nuclear transfer and intracytoplasmic sperm injection in the presence or absence of caffeine or dithiothreitol (DTT) were examined. The following results were found: (1) ovine oocytes were fully aged 30 h post-onset of maturation culture; (2) the appropriate concentrations of caffeine and DTT for oocyte culture were 5 mM and 1 mM, respectively; (3) when nuclear transfer-reconstructed embryos were treated with caffeine or DTT following fusion, no increase in the frequency of development to blastocyst was observed (P > 0.05), but the cell numbers of blastocysts increased (P < 0.05); (4) both caffeine and DTT increased the blastocyst formation rates of intracytoplasmic sperm-injected embryos (P < 0.05); (5) caffeine increased the glutathione content of aged oocytes (P < 0.05). The glutathione content of DTT-treated aged oocytes was higher than that of oocytes matured for 36 h (P < 0.05). In conclusion, caffeine and dithiothreitol delay oocyte ageing but only to a limited extent.

Alteration of sheep coat color pattern by disruption of ASIP gene via CRISPR Cas9.

Coat color is an important characteristic and economic trait in domestic sheep. Aiming at alteration of Chinese merino sheep coat color by genome manipulation, we disrupted sheep agouti signaling protein gene by CRISPR/Cas9. A total of seven indels were identified in 5 of 6 born lambs. Each targeted lamb happened at least two kinds of modifications, and targeted lambs with multiple modifications displayed variety of coat color patterns. Three lambs with 4 bp deletion showed badgerface with black body coat color in two lambs, and brown coat color with light ventral pigmentation in another one. The black-white spotted color was observed in two lambs with 2 bp deletion. Further analysis unraveled that modifications happened in one or more than two copies of ASIP gene, and moreover, the additional spontaneous mutations of D9 and/or D5 preceding the targeting modification could also involve the formation of coat color patterns. Taken together, the entanglement of ASIP modifications by CRISPR/Cas9, spontaneous D9/D5 mutations, and ASIP gene duplications contributed to the variety of coat color patterns in targeted lambs.

iTRAQ-based proteomic profiling of granulosa cells from lamb and ewe after superstimulation

The number of oocytes obtained from lambs after FSH treatment is far greater than those acquired from adult ewes. However, these oocytes typically have reduced viability in comparison with adult ewe oocytes. However, the molecular mechanisms of differences in viability between lamb and ewe oocytes remain unknown. In the present research, we applied iTRAQ coupled with LC-MS/MS proteomic analysis in order to investigate the proteomic expression profile of granulosa cells from lambs and ewes following stimulation with FSH. We detected 5649 proteins; 574 were differentially expressed between adults and juveniles. Based on Gene Ontology enrichment and KEGG pathway analysis, the majority of DEPs are participated in metabolic processes, ribosome and MAPK signaling pathways. Expression levels in ewes turned out to be lower than lambs. Protein interaction network analysis generated by STRING identified MAPK1, SMAD2, SMAD4, CDK1, FOS and ATM as the major findings among 54 significant differentially expressed of proteins. Quantitative real-time PCR analysis was applied to verify the proteomic analysis. These proteins which were identified in lambs may contribute to the reduction of oocyte quality compared to adults. The present research provides understanding of the molecular mechanism for follicle development in lambs.

CRISPR/Cas9‐mediated loss of FGF5 function increases wool staple length in sheep

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.