Increasing the efficiency of homologous recombination vector-mediated end joining repair by inhibition of Lig4 gene using siRNA in sheep embryo fibroblasts.

In animal cells, inhibition of non-homologous end joining (NHEJ) pathway improves the efficiency of homologous recombination (HR)-mediated double-strand brakes (DSBs) repair. To improve the efficiency of HR in sheep embryo fibroblasts, the NHEJ key molecule DNA ligase 4 (Lig4) was suppressed by siRNA interference. Four pairs of siRNA targeting Lig4 were designed and chemically synthesized. These siRNA were electro-transferred into sheep embryo fibroblasts respectively. Compared with the control groups, two pairs of siRNA were identified to effectively inhibit the expression of sheep Lig4 gene by qRT-PCR and Western blotting. The plasmid rejoining assay was adopted for examining the efficiency of HR-mediated DSB repair. I-SceⅠ endonuclease linearized vector and siRNA were co-transfected into sheep embryo fibroblasts. Flow cytometry analysis of cells after transfection for 72 h showed that suppression of Lig4 using siRNAs increased the rejoining efficiency of HR vector by 3-4 times compared with the control groups. Therefore, enhanced HR vector rejoining frequency by instant inhabition of Lig4 gene provides theoretical basis for improving gene targeting efficiency of sheep embryo fibroblasts.

[Expression of Myogenin and MCK genes regulated by PI3K/AKT pathway].

Many intracellular signaling pathways regulate skeletal muscle differentiation. Among them, PI3K/AKT pathway plays an important role. But the mechanisms of chromatin regulation remain unclear. In this study, the murine C2C12 myoblast cell line was used to investigate the expression of Myogenin and MCK genes during the skeletal muscle differentiation. Western blotting analysis showed that the expression of Myogenin and MCK protein was increased significantly after PI3K/AKT activator treatment for 24 h during the C2C12 cell differentiation and the expression of H3K27me3 demethylase UTX was also increased. Chromatin immunoprecipitation (ChIP) and quantitative PCR (Q-PCR) analysis showed that the enrichment of H3K27me3 on the promoter regions of Myogenin and MCK genes and the enhancer region of MCK gene were decreased. It was opposite to the PI3K/AKT inhibitor treatment. We concluded that the PI3K/AKT pathway maybe regulate skeletal muscle differentiation by regulating the expression of UTX gene to change the enrichment of H3K27me3 on the target genes.

[Correlation analysis between polymorphism of the 59383635th locus on X chromosome and fat-tail trait in sheep].

Fat tail or fat rump is one of essential traits for surviving in harsh environments, and the mechanism of fat deposition and its inheritable characters in sheep are still unclear. Therefore, the 59383635th locus on X chromosome in our unpublished chip data was chosen as candidate SNP, PCR-SSCP method was used to detect genotypes in five sheep breeds which have extreme differences in tail types (Altay, Small Tail Han Sheep, Hu, Chinese Merino and Suffolk), and the mathematical model was employed to analyze the correlation between the polymorphism and the trait of fat tail or fat rump. The results in this study showed that the high frequency of allele T exists in Altay flock, and the frequency of allele C appears to be particularly high in the thin tail sheep breeds. The result of mathematical model showed that the ratio of T/C increased exponentially with the increase of phenotype score. These results suggest that there is a big difference in the SNP distribution between fat tail (rump) and thin tail sheep populations, and the SNP can be used as an ideal molecular marker in high-fat or low-fat sheep breeding. However, the biological function of the SNP remains to be further studied.

[Detection and analysis of polymorphisms of 59571364 and 59912586 loci on X chromosome in fat-tail and thin-tail sheep flocks].

In order to analyze the correlation of tail fat deposition and two SNP loci on Ovis arise chromosome X and provide a theoretical basis for using molecular assisted selection technology in further low-fat sheep breeding, the breeds with extreme differences in tail types (Altay, Small Tail Han Sheep, Hu, Chinese Merino and Suffolk) were used to detect the polymorphisms of two SNP loci on X chromosome and the haplotypes with PCR-RFLP method. The results showed that the TT genotype at 59571364 locus and GG genotype at 59912586 locus were preponderant genotypes in thin-tailed Chinese Merino and Suffolk sheep flocks, while the percentage of the two genotypes in fat-tailed (fat-rmup) Altay and Hu flocks is less than 2%. Haplotype analysis showed that CA haplotype is the main haplotype, the percentage of CA is up to 55%, and the percentage of CA and TA haplotypes together was 88.33% in Altay sheep flock. These results suggest that there are great differences in the SNP distribution of the 59571364 and 59912586 loci among different tail-typed sheep flocks, which can be used as molecular markers in high or low fat sheep breeding.

[shRNAs driven by K14 promoter induce tissue-specific RNA interference].

RNA interference is an efficient method for exploring gene function. Accumulating evidence suggests that RNA Pol II promoters can direct cell- or tissue-specific gene silencing. A eGFP-shRNA fusion construct transcribed from an RNA Pol II promoter (K14 promoter) was used to induce gene-specific shRNA silencing ofBMP4 gene expression. Recombinant vectors (pEGFP-C1-shRNA, psiCHECK-BMP4, and pEGFP-K14-shRNA) were constructed. Vectors pEGFP-C1-shRNA and psiCHECK-BMP4 were cotransfected into Hela cells (in vitro) and shRNA-induced inhibition efficiency was tested by a luciferase assay. The results showed that all the six interference sequences inhibited the expression of BMP4 with high efficiency (>60%), and the interference sequence 5# showed the highest efficiency. For in vivo screening of JB6-C41 cells transfected with vector pEGFP-K14-shRNA, the inhibition efficiency was assayed by quantitative RT-PCR and Western blotting analyses. The results showed that the mRNA and protein products of the exogenous BMP4 gene were efficiently and specifically inhibited. The efficiency of gene silencing was greater than 60%, except for sequence 3#. The declines in mRNA and protein expression levels were significantly correlated during gene silence by the shRNA. This system may be adapted for in vivo shRNA expression and gene silencing. This method may provide a novel approach for the application of RNAi technology in suppressing gene expression in the analysis of the mechanisms of hair follicle development in sheep.

[Research progress on genetic diversity of sheep PRNP and resistance breeding].

Prion protein (PrP) is a pathogeny identified in recent years, which infects both mankind and other mammals. It has been proved that PrP is a sole protein able to duplicate and propagate with itself. PrP can express in many tissues and has important physiological functions in many species of animals. The conformation change of PrP is the origin of transmissible spongiform encephalopathies (TSEs). It has been proved that the sheep genetic diversity of prion protein gene (PRNP) is significantly associated with the resistance to scrapie. In this review, the evidence of association between polymorphisms of PRNP and resistance or susceptibility to scrapie and its effects on reproduction and performance traits were focused. The aim is to provide theory guidance for sheep breeding resistant to disease.

Effects of SOF and CR1 media on developmental competence and cell apoptosis of ovine in vitro fertilization embryos.

The present study was to investigate effects of synthetic oviductal fluid (SOF) and Charles Rosenkrans medium (CR1) culture systems on developmental competence and cell apoptosis of ovine in vitro fertilization (IVF) embryos. Ovine presumptive IVF zygotes were cultured in the following six media: (1) SOF supplemented with amino acids (SOFaa) and 8 mg/ml bovine serum albumin (BSA) for 9 days (SOFaaBSA); (2) SOFaa supplemented with 10% fetal bovine serum (FBS) for 9 days (SOFaaFBS); (3) SOFaaBSA for first 3 days and then SOFaaFBS for later 6 days (SOFaaBSA-FBS); (4) CR1 supplemented with amino acids (CR1aa) and 8 mg/ml BSA for 9 days (CR1aaBSA); (5) CR1aa supplemented with 10% FBS for 9 days (CR1aaFBS); (6) CR1aaBSA for first 3 days and then CR1aaFBS for later 6 days (CR1aaBSA-FBS). The rates of blastocyst and hatched blastocyst in group 1, group 3 and group 6 were not different (P>0.05), but were greater than in other three groups (P<0.05). In SOF and CR1 cultural system, SOFaaBSA and CR1aaBSA-FBS provided the highest blastocyst rates respectively. Both numbers of total cell and trophectoderm (TE) in expanded or hatched blastocyst from SOFaaBSA were significantly higher than CR1aaBSA-FBS (P<0.05). However, the inner cell mass (ICM) cell number and ratio of ICM to TE cell in expanded or hatched blastocysts were not different between two groups (P>0.05). The apoptotic signals were firstly observed at 8-cell stage in two groups and became stronger and stronger with the development of embryos. Rates of embryos with apoptotic signals in group 6 at morula or blastocyst were greater than in group 1 (P<0.05). The apoptotic nuclei numbers of morula or blastocyst in group 6 were also significantly higher than group 1 (P<0.05). It is concluded that CR1aaBSA-FBS can support in vitro development of ovine IVF embryos, but SOFaaBSA is more suitable.

[A novel polymorphism of IGFBP-3 gene and its relationship with several wool traits in Chinese Merino sheep].

In this study, PCR-SSCP analysis was used to identify genetic variation in IGFBP-3 gene in Chinese Merino and Kazakh sheep. A PCR product of 178 bp corresponding to partial intron1 illustrated three unique binding patterns by SSCP analysis. Frequencies of the genotype AA, AB, BB and allele A, B in Chinese Merino sheep were 0.70, 0.24, 0.06, and 0.82, 0.18 respectively , and they were 0.87, 0.13, 0.00, and 0.93, 0.07 respectively in Kazaka sheep. Sequence analysis revealed a G/T transversion at position 122 of the fragment. This polymorphic locus of IGFBP-3 gene was at Hardy-Weinberg dis-equilibrium (P<0.01) in the two breeds. Different genotypes slightly affected several wool traits of Chinese Merino sheep. The individuals of genotype AA, AB, and BB had no significant difference in post-shearing weight and clean wool rate. Sta-ple length (SL) was decreased with the genotype of AA, AB, and BB, and the difference between AA and AB was significant (P<0.01). Greasy fleece weight (GFW) and follicle density in individuals of genotype AA was significantly lower than that in individuals of genotype AB (P<0.01) and BB (P<0.05); Average fiber diameter (AFD) in individuals of genotype AA was significantly higher than that in individuals of genotype AB (P<0.01) and BB (P<0.05).

[Relationship between genetic diversity of chromosome 6 determined by microsatellite markers and litter size in Hu sheep].

Genetic diversity and relationship with litter size were analyzed in Hu sheep using 6 microsatellite markers LSCV043, BMS2508, GC101, 300U, Bulge5, and 471U, which were closely linked to FecB gene. Thirty-four alleles and 53 genotypes were detected at 6 microsatellite loci, which were all polymorphic. Three markers LSCV043, BMS2508 and 300 U were highly polymorphic loci. The polymorphism information content (PIC) were 0.6674, 0.6035, and 0.5615, respectively. The total mean of litter size of genotype LSCV043 107 bp/123 bp was significantly higher than that of genotype 110 bp/123 bp. The litter size of the first lambing for genotypes BMS2508 154 bp/154 bp, 154 bp/170 bp and 154 bp/200 bp were significantly higher than that of genotype 170 bp/170 bp (P<0.05). Furthermore, total litter size for BMS2508 170 bp/170 bp was the lowest in all the genotypes within this locus (P<0.05). No significant difference was detected in other genotypes.

[The preparation of oligonucleotide chips for detecting BMPR-IB gene polymorphism in sheep].

BMPR-IB gene increases ovulation rate and litter size as a major gene in Chinese Merino, because of 746 A to G mutation. The aim of this study was to make oligonucleotide chips to determine single nucleotide polymorphism (SNP) in BMPR-IB gene. The oligonucleotide chips were manufactured by using six sequence-specific oligonucleotide probes derived from polymorphic regions in the mutation and spotting the probes by microarrayer onto the aldehyde modified glass slides. The 746 A to G mutation was detected with ovine blood in reaction cells of chips. The results were identified by designed the Arraydoctor 2.0 software, in accordance with those of restriction fragment length polymorphisms (RFLP). The results showed the oligonucleotide chips are a parallel, accurate and effective way for screening prolificacy in molecular-assisted selection(MAS)of sheep.

Polymorphism of FecB gene in nine sheep breeds or strains and its effects on litter size, lamb growth and development.

Nine sheep breeds or strains, including 615 individuals were screened with forced PCR RFLP method for the FecB gene to study the polymorphism and its effects on litter sizes, body weights and body sizes. Results show that the polymorphism frequencies of FecB gene are significantly imbalanced in these breeds or strains. The Hu sheep were all homozygous carriers (BB). In the Chinese Merino prolific meat strain, the genotype frequencies of BB, B+ and ++ are 51%, 30% and 19%, respectively, whereas all the other flocks had only the wild-type (++) genotype. Results within Chinese Merino prolific meat strain showed that mean litter sizes of ewes with genotype BB and B+ are 2.8 (+/-0.74) and 2.3 (+/-0.63) (P > 0.05), whereas ++ ewes had a litter size of only 1.2 (+/-0.68) (P < 0.01). At 90 days after birth, the body weights of BB/B+ lambs were higher than that of ++ lambs (18.6 +/- 3.70 kg, 18.0 +/- 3.71 kg versus 15.6 +/- 2.22 kg, P < 0.05). In addition, the heart girth and chest width of BB/B+ lambs were significantly longer than ++ lambs (P < 0.05). No significant differences were observed in either body weight or body size at day 120. Litter size at first lambing from Hu at Natural Source Conservative Region was found to be significantly higher than that from the other two regions sampled (P < 0.05). In addition to the additive effect on litter size, these findings show for the first time that the FecB gene had a positive effect on early postnatal body growth.

[Relationship between microsatellite DNA polymorphism and hind-quarter development in sheep chromosome 18].

Ten highly polymorphic microsatellite loci possibly linked to or correlated with the Callipyge gene were selected, according to the genetic map and linkage map of sheep chromosome 18. They were ILSTS54, TGLA337, HH47, TGLA122, BP33, OB2, BM3413, MCM38, MCMA26 and CSSM18. Polymorphisms of these microsatellites were detected in 61 Dorset (male) x Xinjiang fine wool sheep (female) samples and 76 Suffolk (male) x Xinjiang fine wool sheep (female) samples. Results showed that the number of alleles for the 10 microsatellite loci, heterozygosity and PIC (polymorphism information content) in the Dorset population were 8-16, 0.8370-0.9252, and 0.8221-0.9167, respectively. The same parameters in the Suffolk population were 5-10, 0.7603-0.8913 and 0.7176-0.8809, respectively. The effect of these loci on hindquarter width was analyzed in a generalized linear model. Results showed that, in the Dorset group, BM3413, MCMA26 and CSSM18 each had a significant effect on hindquarter width (P<0.05), while the other seven loci had no effect on it (P>0.05). In the Suffolk group, TGLA122, BM3413, MCM38 and CSSM18 had a significant effect on hindquarter width (P<0.05), while the other six loci did not (P>0.05). Our results also indicated that the cause of hindquarters hypertrophy in Xinjiang meat sheep may be different from the A-to-G mutation between the region of DLK1 and GTL2 . There may be other genes or QTL (quantitative trait loci) that affect hindquarter muscle development on chromosome 18 in Xinjiang meat sheep.

The tissue distribution and developmental changes of ghrelin mRNA expression in sheep.

Male Kazak sheep and Xinjiang fine wool sheep, six for each different age group (days 2, 30, 60, 90 and 120), were used in the present study to investigate the tissue distribution and developmental changes of ghrelin mRNA expression in abomasum; however, there was no 120-day-old Kazak sheep. After measurement of body weight, the tissues such as hypothalamus, pituitary, heart, liver, rumen, reticulum, omasum, abomasum, duodenum, and longissimus dorsi muscle were sampled. And the total RNA of different tissues was extracted to determine the abundance of ghrelin mRNA by RT-PCR and real-time PCR. The results showed that (1) for both breeds, body weight among different ages was significantly different (P<0.05). And from day 30 to 90, the body weight of Kazak was significantly higher than that of Xinjiang (P<0.01); (2) Ghrelin mRNA existed in all the above tissues and was significantly higher in the abomasum than in other tissues (P<0.05); (3) the temporal patterns of abomasum ghrelin mRNA expression in Kazak and Xinjiang were similar. From day 2 to 60 in Kazak and 2 to 90 in Xinjiang, there was a steady increase in the ghrelin mRNA level. By day 60 in Kazak and day 90 in Xinjiang, the level reached a plateau and remained steady. These results also demonstrated that from birth to day 90, ghrelin mRNA level was significantly higher in Kazak than in Xinjiang (P<0.01).

[Polymorphism analysis of genes associated with hindquarters muscular development on chromosome 18 in Xinjiang meat sheep].

Through polymorphism analysis of genes associated with hindquarters development on chromosome 18 in Xinjiang meat sheep, we sought genes that are associated with increased hindquarters musculature in the hope to provide theoretical guidance to molecular marker-assisted selection of meat sheep. The polymorphism of Callipyge (CLPG) gene was analyzed by PCR-SSCP and PCR-RFLP in populations of Dorset and Suffolk breeds and their F1 and F2 crosses with local Xinjiang Fine Wool sheep. Results showed that there was no PCR-RFLP polymorphism at 103849 bp in the region between DLK1 and GTL2 on chromosome 18, which suggested that hindquarters over-development was not controlled by the callipyge gene (CLPG) in Xinjiang meat sheep group. In addition, single nucleotide polymorphisms (SNPs) were detected at the Meg3.9 locus on chromosome 18 by PCR-SSCP. Based on the polymorphism, three genotypes AA, AB and AC could be discerned in this locus, with AA being the major one. The relationship between the genotypes and hindquarters hypertrophy was also investigated in the same group. Results indicated that the AC genotype had a significant impact on the hindquarters hypertrophy while the other two were not related to the muscling trait. In F1 crosses between Dorset and local fine wool sheep breeds, there was a significant difference in carcass weight and slaughter rate between the AC genotype and the other two genotypes (P<0.05), but there was not significant difference in the monthly live weight among the three genotypes (P<0.05). Based on the above, we found the muscling trait did not result from the mutation in at the 9571-268.3 site of the CLPG locus. Thus it may be under the influence of other genes or multiple linked QTL.

The developmental changes and effect on IMF content of H-FABP and PPARgamma mRNA expression in sheep muscle.

Male Kazak sheep and Xinjiang fine wool sheep of different ages were selected to investigate the developmental changes and effect on intramuscular fat (IMF) content of heart fatty acid-binding protein (H-FABP) and peroxisome proliferator-activated receptor gamma (PPARgamma) mRNA expression in muscle. Longissimus dorsal muscle was sampled to measure IMF content; and total RNA was extracted to determine H-FABP and PPARgamma mRNA expression levels by real-time PCR. The results showed that: (1) The IMF content increased continuously with growing and showed significant differences (P<0.05) between ages in male Kazak sheep, but no such differences (P>0.05) existed in Xinjiang fine wool sheep. Furthermore, the IMF content in Kazak sheep was very much higher (P<0.01) than that of the other breed from day 30 to 90; (2) H-FABP mRNA expression level was the highest on day 2 and showed significant differences (P<0.05) between ages in male Kazak sheep as well as in Xinjiang fine wool sheep. In the former breed, the expression reached the lowest point at day 30, and then rose continuously. But in the latter breed, it declined continuously from day 2 to 90, and then increased; (3) Significant differences (P<0.05) of PPARgamma mRNA expression between ages occurred in both breeds. In male Kazak sheep, PPARgamma mRNA expression declined from day 2 to 90, while in the other breed it increased continuously from day 2 to 60, but reached the lowest level at day 90, then increased; (4) In male Kazak sheep, the mRNA expression level of H-FABP was highly positively correlated (r=0.737, P<0.01) with IMF content from day 30 to 90, but that of PPARgamma was highly negatively correlated (r=-0.835, P<0.01) with IMF content from day 2 to 90.

Polymorphism of FecB gene in nine sheep breeds or strains and its effects on litter size, lamb growth and development.

Nine sheep breeds or strains, including 615 individuals were screened with forced PCR RFLP method for the FecB gene to study the polymorphism and its effects on litter size, body weight and body size. Results showed that the polymorphism frequencies of FecB gene were significantly imbalanced in these breeds or strains. The Hu sheep were all homozygous carriers of FecB gene(BB). In the Chinese Merino prolific meat strain, the genotype frequencies of BB, B+ and ++ were 51%, 30% and 19%, respectively, whereas all the other flocks had only the wild-type (++) genotype. Results within the Chinese Merino prolific meat strain showed that the mean litter size of ewes with genotype BB and B+ were 2.8 (+/-0.74) and 2.3 (+/- 0.63) (P < 0.05), whereas ++ genotype ewes had a litter size of only 1.2 (+/-0.68) (P < 0.01). At day 90 after birth, the body weights of BB/B+ genotype lambs were higher than that of ++ genotype lambs (18.6 +/- 3.70 kg, 18.0 +/- 3.71 kg vs 15.6 +/- 2.22 kg, P < 0.05). In addition, the heart girth and chest width of BB/B+ genotype lambs were significantly longer than those of the ++ lambs (P < 0.05). No significant differences were observed in either body weight or body size at day 120. Litter size at first lambing from Hu at Natural Source Conservative Region was found to be significantly higher than that from the other two regions sampled (P < 0.05). In addition to the additive effect on litter size, these findings showed for the first time that the FecB gene had a positive effect on early postnatal body growth.

[Development of a rapid mismatch PCR method using tetra-primer ARMS for detection of BMPR-IB gene mutation in sheep].

Tetra-primer ARMS PCR is a rapid, simple and efficient method for the detection of single nucleotide polymorphism (SNP). Bone morphogenetic protein receptor IB (BMPR-IB) gene is a major effect gene that controls prolific trait in Booroola Merino sheep. The aim of this study was to establish a tetra-primer ARMS PCR method for the detection of BMPR-IB genotype. Specific primers around the mutation locus (A746G) were designed according to the principle of tetra-primer amplification refractory mutation system PCR. Furthermore, a pair of primers were designed to amplify the DNA fragment, which contain the mutation locus as positive control, and an individual genotype was directly detected by one round of PCR amplification. The detection efficiency of sheep BMPR-IB genotyping was greatly improved by use of this method.

[Detection of major gene on litter size in sheep].

The current study was designed to detect SNPs within BMP15 and BMPR-IB gene and investigate the effect of the genes on sheep litter size. Four sheep lines, HU-Yang, Chinese Merino monotocous, Chinese Merino multiparous for wool production and Chinese Merino multiparous for mutton production, were used in this study. Litter sizes were recorded for each ewe in the four lines. Primers for BMP15 and BMPR-IB gene were designed from database sheep sequence and polymorphisms were detected by PCR-RFLP method. The results showed that there was no polymorphism with BMP15 gene among the four lines, and there was an A / G SNP with BMPR-IB gene at base 746 among the four lines. Three types of genotype (BB, B+ and ++), based on A / G locus, were found within each line. The frequencies of genotypes were significantly different among the lines (P<0.001), with BB genotype primarily existing in HU-Yang, ++ genotype in Chinese Merino monotocous line, and B+ genotype in Chinnese Merino multiparous lines. The A / G mutation influence significantly the sheep litter sizes, and the BB and B+ ewes had significant higher litter sizes than ++ ewes. The results of present study showed simultaneously that the genotype of BMPR-IB was a perfect predictor of the sheep litter sizes. These results intensively indicated that BMPR-IB is a major gene to affect litter size in sheep, and could be used as the molecular genetic marker to select litter size in sheep.