Myostatin (MSTN), associated with the “double muscling” phenotype, affects muscle growth and fat deposition in animals, whereas how MSTN affects adipogenesis remains to be discovered. Here we show that MSTN can act through the MEF2C/miR222/SCD5 cascade to regulate fatty acid metabolism. We generated MSTN-knockout (KO) cloned Meishan pigs, which exhibits typical double muscling trait. We then sequenced transcriptome of subcutaneous fat tissues of wild-type (WT) and MSTN-KO pigs, and intersected the differentially expressed mRNAs and miRNAs to predict that stearoyl-CoA desaturase 5 (SCD5) is targeted by miR222. Transcription factor binding prediction showed that myogenic transcription factor 2C (MEF2C) potentially binds to the miR222 promoter. We hypothesized that MSTN-KO upregulates MEF2C and consequently increases the miR222 expression, which in turn targets SCD5 to suppress its translation. Biochemical, molecular and cellular experiments verified the existence of the cascade. This novel molecular pathway sheds light on new targets for genetic improvements in pigs.

In mammalian cells, double-strand breaks (DSBs) are repaired predominantly by error-prone non-homologous end joining (NHEJ), but less prevalently by error-free template-dependent homologous recombination (HR). DSB repair pathway selection is the bedrock for genome editing. NHEJ results in random mutations when repairing DSB, while HR induces high-fidelity sequence-specific variations, but with an undesirable low efficiency. In this review, we first discuss the latest insights into the action mode of NHEJ and HR in a panoramic view. We then propose the future direction of genome editing by virtue of these advancements. We suggest that by switching NHEJ to HR, full fidelity genome editing and robust gene knock-in could be enabled. We also envision that RNA molecules could be repurposed by RNA-templated DSB repair to mediate precise genetic editing.

ABSTACT Myostatin (MSTN) resulted in reduced backfat thickness in MSTN-knockout (MSTN-KO) pigs, whereas the underlying mechanism remains elusive. In this study, RNA sequencing (RNA-seq) was used to screen differentially expressed genes (DEGs) in porcine fat tissues. We identified 285 DEGs, including 4 adipocyte differentiation-related genes (ADRGs). Matrix Metalloproteinase-2/7 (MMP-2/7), fibronectin (FN), and laminin (LN) were differentially expressed in MSTN-KO pigs compared with wild-type (WT) pigs. To investigate the molecular mechanism, we treated the preadipocytes with siRNA and recombinant MSTN protein. The results indicated that MSTN increased the expression of MMP-2/7/9 and promoted the preadipocyte differentiation. To further validate the effect of MSTN on MMP-2/7/9 expression, we treated MSTN-KO PK15 cells with recombinant MSTN protein and detected the expression of MMP-2/7/9. The data showed that MSTN increases the expression of MMP-2/7/9 in PK15. This study revealed that MSTN promoted preadipocyte differentiation and provided the basis for the mechanism of fatty deposition in pigs.

Feeder cells can promote cell proliferation and help overcome the developmental arrest of early embryos by producing growth factors. The objective of this study was to evaluate the effects of feeder cells on the development of all single porcine parthenogenetic embryos in vitro. Firstly, we showed that the cleavage and blastocyst formation rate of all single procine parthenogenetic embryos co-cultured with feeder cells increased in contrast to those cultured without feeder cells (p0.05). Secondly, highly significant differences were observed between the cleavage and blastocyst formation rate (p<0.05) when the embryos were co-cultured with oviduct epithelial feeder cells in different volume drops ranging from 3 to 20 mu L and the cleavage rate were the highest when cultured in 5 mu L drops. Thirdly, the tempospacial pattern of the development of single embryos co-cultured with oviduct epithelial feeder cells was consistent with that of traditional multi-embryo culture, indicating that the co-culturing does not affect the developmental competence of the porcine parthenogenetic embryos. Finally, highly significant diffe-rences were observed between the cleavage and blastocyst formation rate with and without zona pellucida in vitro (p<0.05). In this study, a new adaption of in vitro co-culture of single porcine parthenogenetic embryos using feeder cells has been successfully established and this will facilitate further investigations to discover the mechanistic mode of developmental arrest of porcine embryos.