Qi-Rong Luo

Quisinostat treatment improves histone acetylation and developmental competence of porcine somatic cell nuclear transfer embryos.

Abnormal epigenetic modifications are considered a main contributing factor to low cloning efficiency. In the present study, we explored the effects of quisinostat, a novel histone deacetylase inhibitor, on blastocyst formation rate in porcine somatic‐cell nuclear transfer (SCNT) embryos, on acetylation of histone H3 lysine 9 (AcH3K9), and on expression of POU5F1 protein and apoptosis‐related genes BAX and BCL2. Our results showed that treatment with 10 nM quisinostat for 24 hr significantly improved the development of reconstructed embryos compared to the untreated group (19.0 ± 1.6% vs. 10.2 ± 0.9%; p < 0.05). Quisinostat‐treated SCNT embryos also possessed significantly increased AcH3K9 at the pseudo‐pronuclear stage (p < 0.05), as well as improved immunostaining intensity for POU5F1 at the blastocyst stage (p < 0.05). While no statistical difference in BAX expression was observed, BCL2 transcript abundance was significantly different in the quisinostat‐treated compared to the untreated control group. Of the 457 quisinostat‐treated cloned embryos transferred into three surrogates, six fetuses developed from the one sow that became pregnant. These findings suggested that quisinostat can regulate gene expression and epigenetic modification, facilitating nuclear reprogramming, and subsequently improving the developmental competence of pig SCNT embryos and blastocyst quality.

Generation of cloned adult muscular pigs with myostatin gene mutation by genetic engineering

Skeletal muscle is the most economically valuable tissue in meat-producing animals and enhancing muscle growth in these species may enhance the efficiency of meat production. Skeletal muscle mass is negatively regulated by myostatin (MSTN), and non-functional mutations of the MSTN gene in various animal species have led to dramatic hypermuscularity. This study was designed to assess the characteristics of male MSTN-knockout (KO) pigs. A transcription activator-like effector nuclease (TALEN) pair targeting exon 1 of the swine MSTN gene was constructed and used to transfect porcine fetal fibroblasts (PFFs). We obtained a cell line consisting of a 2-bp deletion in one allele and a 4-bp deletion in the other allele, and this was used as a donor to generate cloned pigs via SCNT, and delivered 18 live piglets. They developed and grew normally to sexual maturity. These MSTN-KO boars grew normally to adulthood and showed visually-clear hypermuscular characteristics, increased carcass dressing percentage and loin eye size, and decreased backfat thickness. These pigs may show greater meat production, as well as being used in animal models of human diseases.

Histone deacetylase inhibitor M344 significantly improves nuclear reprogramming, blastocyst quality, and in vitro developmental capacity of cloned pig embryos

M344 is a novel histone deacetylase inhibitor. There is no report on the effect of M344 treatment on the development of pig embryos after somatic cell nuclear transfer (SCNT). In the present study, we investigated the effect of M344 on the blastocyst formation rate in cloned embryos, acetylation level of histone H4 lysine 12 (AcH4K12), and the expression of pluripotency-related genes , , and . Our results indicated that treatment with 5 µ M344 for 6 h improved the development of porcine embryos, in comparison with the untreated group (25.1% ± 5.0 vs. 10.9% ± 2.4; < 0.05). Moreover, M344-treated embryos had increased average fluorescence intensity of AcH4K12 at the pseudo-pronuclear stage ( < 0.05). However, no differences exist in Oct4, NANOG, and SOX2 expression in M344-treated and untreated SCNT blastocysts. In evaluating the effect of M344 on in vivo development, 845 M344-treated embryos were transferred into 3 surrogates, 1 of whom became pregnant and developed 3 fetuses. These findings suggested that M344 elevated the level of histone acetylation, facilitated the nuclear programming, and subsequently improved the developmental competence of pig SCNT embryos.

The cyclin-dependent kinase inhibitor, JNJ-7706621, improves in vitro developmental competence of porcine parthenogenetic activation and somatic cell nuclear transfer embryos

In this study we examined the effects of JNJ-7706621, a cyclin-dependent kinase inhibitor, on the in vitro growth of pig embryos that had been produced either by parthenogenetic activation (PA) or somatic cell nuclear transfer (SCNT). A significantly higher percentage of PA embryos reached the blastocyst stage by Day 7 after exposure to 10µM JNJ-7706621 for 4h compared with embryos exposed to 5µgmL-1 cytochalasin B for 4h (P<0.05). Similarly, the rate of Tyr15 phosphorylation of the complex of cyclin and p34cdc2 (CDK1) was significantly elevated in the JNJ-7706621-treated embryos compared with embryos exposed to cytochalasin B or non-treated controls (P<0.05). In contrast, Thr161 phosphorylation of CDK1 was significantly lower in the JNJ-7706621-treated group compared with the cytochalasin B-treated as well as the non-treated group (P<0.05). Similarly, the level of M-phase-promoting factor (MPF) in embryos was significantly lower in the JNJ-7706621-treated group compared with the cytochalasin B-treated and non-treated groups (P<0.05). In addition, more SCNT embryos reached the blastocyst stage after treatment with JNJ-7706621 than following exposure to cytochalasin B (P<0.05). In conclusion, these results reveal that exposure to 10µM JNJ-7706621 for 4h improves early development of PA and SCNT porcine embryos by suppressing the activity of CDK1 and a concomitant reduction in the level of MPF.

Cotreatment with RepSox and LBH589 improves the in vitro developmental competence of porcine somatic cell nuclear transfer embryos

Accumulating evidence suggests that aberrant epigenetic reprogramming and low pluripotency of donor nuclei lead to abnormal development of cloned embryos and underlie the inefficiency of mammalian somatic cell nuclear transfer (SCNT). The present study demonstrates that treatment with the small molecule RepSox alone upregulates the expression of pluripotency-related genes in porcine SCNT embryos. Treatment with the histone deacetylase inhibitor LBH589 significantly increased the blastocyst formation rate, whereas treatment with RepSox did not. Cotreatment with 12.5μM RepSox and 50nM LBH589 (RepSox+LBH589) for 24h significantly increased the blastocyst formation rate compared with that of untreated embryos (26.9% vs 8.5% respectively; P<0.05). Furthermore, the expression of pluripotency-related genes octamer-binding transcription factor 4 (NANOG) and SRY (sex determining region Y)-box 2 (SOX2) were found to significantly increased in the RepSox+LBH589 compared with control group at both the 4-cell and blastocyst stages. In particular, the expression of NANOG was 135-fold higher at the blastocyst stage in the RepSox+LBH589 group. Moreover, RepSox+LBH589 improved epigenetic reprogramming. In summary, RepSox+LBH589 increases the expression of developmentally important genes, optimises epigenetic reprogramming and improves the invitro development of porcine SCNT embryos.

Parthenogenetic activation and somatic cell nuclear transfer of porcine oocytes activated by an electric pulse and AZD5438 treatment

We examined the in vitro developmental competence of parthenogenetic activation (PA) oocytes activated by an electric pulse (EP) and treated with various concentrations of AZD5438 for 4 h. Treatment with 10 µM AZD5438 for 4 h significantly improved the blastocyst formation rate of PA oocytes in comparison with 0, 20, or 50 µM AZD5438 treatment (46.4% vs. 34.5%, 32.3%, and 24.0%, respectively; P 0.05). Furthermore, 66.67% of blastocysts derived from these AZD5438-treated PA oocytes had a diploid karyotype. The blastocyst formation rate of PA and somatic cell nuclear transfer (SCNT) embryos was similar between oocytes activated by an EP and treated with 2 mM 6-dimethylaminopurine for 4 h and those activated by an EP and treated with 10 µM AZD5438 for 4 h (11.11% vs. 13.40%, P > 0.05). In addition, the level of maturation-promoting factor (MPF) was significantly decreased in oocytes activated by an EP and treated with 10 µM AZD5438 for 4 h. Finally, the mRNA expression levels of apoptosis-related genes (Bax and Bcl-2) and pluripotency-related genes (Oct4, Nanog, and Sox2) were checked by RT-PCR; however, there were no differences between the AZD5438-treated and non-treated control groups. Our results demonstrate that porcine oocyte activation via an EP in combination with AZD5438 treatment can lead to a high blastocyst formation rate in PA and SCNT experiments.