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The technique of pig cloning holds great promise for the livestock industry, life science, and biomedicine. However, the prenatal death rate of cloned pig embryos is extremely high, resulting in a very low cloning efficiency. This limits the development and application of pig cloning. In this study, we utilized embryo biopsy combined with microproteomics to identify potential factors causing the developmental arrest in cloned pig embryos. We verified the roles of two potential regulators, PDCD6 and PLK1, in cloned pig embryo development. We found that siRNA-mediated knockdown of PDCD6 reduced mRNA and protein expression levels of the pro-apoptotic gene, CASP3, in cloned pig embryos. PDCD6 knockdown also increased the cleavage rate and blastocyst rate of cloned porcine embryos. Overexpression of PLK1 via mRNA microinjection also improved the cleavage rate of cloned pig embryos. This study provided a new strategy to identify key factors responsible for the developmental defects in cloned pig embryos. It also helped establish new methods to improve pig cloning efficiency, specifically by correcting the expression pattern of PDCD6 and PLK1 in cloned pig embryos. [ABSTRACT FROM AUTHOR]

The technique of cloning has wide applications in animal husbandry and human biomedicine. However, the very low developmental efficiency of cloned embryos limits the application of cloning. Ectopic XIST-expression-induced abnormal X chromosome inactivation (XCI) is a primary cause of the low developmental competence of cloned mouse and pig embryos. Knockout or knockdown of XIST improves cloning efficiency in both pigs and mice. The transcription factor Yin yang 1(YY1) plays a critical role in XCI by triggering the transcription of X-inactive specific transcript (XIST) and facilitating the localization of XIST RNA on the X chromosome. This study aimed to investigate whether RNA interference to suppress the expression of YY1 can inhibit erroneous XIST expression, rescue abnormal XCI, and improve the developmental ability of cloned pig embryos. The results showed that YY1 binds to the 5′ regulatory region of the porcine XIST gene in pig cells. The microinjection of YY1 siRNA into cloned pig embryos reduced the transcript abundance of XIST and upregulated the mRNA level of X-linked genes at the 4-cell and blastocyst stages. The siRNA-mediated knockdown of YY1 altered the transcriptome and enhanced the in vitro and in vivo developmental efficiency of cloned porcine embryos. These results suggested that YY1 participates in regulating XIST expression and XCI in cloned pig embryos and that the suppression of YY1 expression can increase the developmental rate of cloned pig embryos. The present study established a new method for improving the efficiency of pig cloning. [ABSTRACT FROM AUTHOR]

Pig cloning technique is valuable in agriculture, biomedicine, and life sciences. However, the full-term developmental efficiency of cloned pig embryos is only about 1%, which limits pig cloning application. The quality of recipient oocytes greatly affects the developmental competence of cloned pig embryos. Thus, this study investigated the effects of a recipient oocyte source (in vivo matured [IVVM] oocytes vs. slaughter house-derived in vitro matured [IVTM] oocytes), and follicular liquid treatment (slaughter house-derived immature follicle-derived fluid [IFF] vs. in vivo-matured follicle-derived fluid [MFF]) during the in vitro maturation (IVM) of oocytes on the development of the cloned pig embryos. Our results showed that using IVVM oocytes to replace IVTM oocytes as recipient oocytes, and using 10% MFF IVM medium to replace 10% IFF IVM medium could enhance the development of the cloned pig embryos. IFF and MFF contained different levels of oocyte quality-related proteins, resulting in different oocyte quality-related gene expression levels and reactive oxygen species levels between the 10% MFF medium-cultured oocytes and 10% IFF medium-cultured oocytes. This study provided useful information for enhancing the pig cloning efficiency by improving the quality of recipient oocytes. [ABSTRACT FROM AUTHOR]