Frameshift mutation in myostatin gene by zinc-finger nucleases results in a significant increase in muscle mass in Meishan sows

Myostatin (MSTN) is a negative regulator of skeletal muscle growth and development. A significant increase in skeletal muscle was observed in Mstn^−/− mice compared with wild-type mice. So far, there has been no report on porcine MSTN mutations leading to skeletal muscle hypertrophy. In this report a MSTN frameshift mutation missing 11 nucleotides in exon 2 was introduced into Meishan pigs by zinc finger nuclease (ZFN) technology. ZFN-edited MSTN^−/− Meishan pigs were successfully produced by a cloning method of somatic cell nucleus transfer. Results from slaughter experiments indicated that lean meat yield increased 16.53% in about 80 kg (10-months-old) MSTN^−/− Meishan sows compared with their corresponding wild-type counterparts. The lean percentage of carcass from MSTN^−/− sows was 61.20% vs 48.25% for MSTN^+/− sows and 44.67% for wild-type sows. The fat of MSTN^−/− sows was significantly lower than that of MSTN^+/− and wild-type sows. The loin eye area of MSTN^−/− Meishan sows (56.42 cm²) was greater than that of MSTN^+/− (37.39 cm²) and wild-type (26.26 cm²) sows. The muscle fibre area of longissimus muscle in wild-type Meishan sows (1 946 μm²) was significantly greater than that of MSTN^+/− (1 324 μm²) and MSTN⁻^/− (1 419 μm²) sows. Moreover the significantly increased skeletal muscle in these MSTN^−/− Meishan sows was mainly due to the increase in the number of myofibres rather than to hypertrophy. Compared with wild-type Meishan sows, it was noted that myofibres had transformed from type I to IIB in MSTN^−/− Meishan sows. Our present study demonstrated that frameshift mutation in MSTN by ZFN technology led to a significant increase in muscle mass and a significant decrease in fat content in Meishan sows.

Cloning of a gene-edited macaque monkey by somatic cell nuclear transfer

Cloning of macaque monkeys by somatic cell nucleus transfer (SCNT) allows the generation of monkeys with uniform genetic backgrounds that are useful for the development of non-human primate models of human diseases. Here, we report the feasibility of this approach by SCNT of fibroblasts from a macaque monkey (Macaca fascicularis), in which a core circadian transcription factor BMAL1 was knocked out by clustered regularly interspaced short palindromic repeat/Cas9 gene editing (see accompanying paper). Out of 325 SCNT embryos transferred into 65 surrogate monkeys, we cloned five macaque monkeys with BMAL1 mutations in both alleles without mosaicism, with nuclear genes identical to that of the fibroblast donor monkey. Further peripheral blood mRNA analysis confirmed the complete absence of the wild-type BMAL1 transcript. This study demonstrates that the SCNT approach could be used to generate cloned monkeys from fibroblasts of a young adult monkeys and paves the way for the development of macaque monkey disease models with uniform genetic backgrounds.

25 THE DEVELOPMENTAL COMPETENCE AND PLURIPOTENT GENE EXPRESSION OF PORCINE SOMATIC CELL NUCLEUS TRANSFER EMBRYOS IMPROVED BY m-CARBOXYCINNAMIC ACID BISHYDROXAMIDE

Differentiated nuclei can experimentally be returned to an undifferentiated embryonic state after nuclear transfer (NT) to unfertilized metaphase II (MII) oocytes. Nuclear reprogramming is triggered immediately after somatic cell nucleus transfer (SCNT) into recipient cytoplasm and this period is regarded as a key stage for optimizing reprogramming. In a recent study (Miyamoto et al. 2010 JBC), use of m-carboxycinnamic acid bishydroxamide (CBHA), a histone deacetylase inhibitor, during the in vitro early culture of murine cloned embryos modifies the acetylation status of somatic nuclei and increases the developmental competence of SCNT embryos. Thus, we examined the effects of CBHA on the in vitro developmental competence and pluripotent gene expression of porcine SCNT embryos. The porcine cloned embryos were treated with a 100 μM concentration of CBHA during the in vitro early culture (10 h) and then were assessed to cleavage rates and development to the blastocyst stage. In addition, pluripotent gene expression of SCNT embryos was analysed by RT-PCR. All data were analysed by chi-square. Following 4 replicates (207 and 124 for NT and CBHA-treated NT embryos, respectively), no significant difference was observed between NT and CBHA-treated NT embryos for cleavage rate (83.9 vs 82.2%). However, the developmental competence to the blastocyst stage differed significantly between NT and CBHA-treated NT embryos (6.8 vs 18.6%; P < 0.05). In addition, pluripotent transcription factors including Oct4, Nanog and Sox2 were higher expressed in the cloned embryos treated with CBHA (P < 0.05). The results of the present study suggest that treatment with CBHA as a histone deacetylase inhibitor significantly increased the developmental competence as well as the pluripotent gene expression of porcine SCNT embryos. This work was partly supported by a grant from the NRF (2011-0013703) and the Next-Generation BioGreen 21 Program (No. PJ008209), Rural Development Administration, Republic of Korea.