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Efficient and precise genetic engineering in livestock such as cattle holds great promise in agriculture and biomedicine. However, techniques that generate pluripotent stem cells, as well as reliable tools for gene targeting in livestock, are still inefficient, and thus not routinely used. Here, we report highly efficient gene targeting in the bovine genome using bovine pluripotent cells and clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 nuclease. First, we generate induced pluripotent stem cells (iPSCs) from bovine somatic fibroblasts by the ectopic expression of yamanaka factors and GSK3β and MEK inhibitor (2i) treatment. We observed that these bovine iPSCs are highly similar to naïve pluripotent stem cells with regard to gene expression and developmental potential in teratomas. Moreover, CRISPR/Cas9 nuclease, which was specific for the bovine NANOG locus, showed highly efficient editing of the bovine genome in bovine iPSCs and embryos. To conclude, CRISPR/Cas9 nuclease-mediated homologous recombination targeting in bovine pluripotent cells is an efficient gene editing method that can be used to generate transgenic livestock in the future.

Abstract. Mitogen-activated protein kinase (MAPK) and maturation/M phase promoting factor (MPF) play crucial roles in oocyte meiotic maturation in mammals. However, the underlying molecular mechanisms have not been addressed. In this study, the effects of the MEK/MAPK pathway inhibitor U0126 and the MPF inhibitor roscovitine on meiotic maturation and maternal gene expression in pig cumulus-oocyte complexes (COCs) and denuded oocytes (DOs) were investigated. Both inhibitors can reversibly block the resumption of meiosis in pig oocytes. COCs or DOs initially cultured in drug-free medium for 24 h and then transferred to medium containing U0126 showed normal progress to the Metaphase II stage (MII); (90.38 vs. 92.16% control group). In contrast, roscovitine treatment from 24 to 44 h significantly inhibited maturation of COCs and DOs. To explore the underlying molecular mechanisms, expression patterns and polyadenylation states of five representative maternal transcripts, cyclin B1, Cdc2, C-mos, GDF9 and BMP15, were examined by real-time PCR and poly(A)-test PCR (PAT assay). U0126 treatment resulted in aberrant expression of Cdc2 and GDF9, while roscovitine significantly maintained all five maternal transcripts at very high levels in treated COCs compared with the control group. The polyadenylation of these mRNAs increased as well. Furthermore, the experiments were repeated in DOs, and the results also indicated that both Cdc2 and GDF9 showed significantly higher expression in both mRNA and polyadenylation levels in the drug treatment groups. Together, these results provide the first demonstration in a mammalian system that MAPK and MPF play important roles in regulation of maternal gene expression during oocyte maturation. Key words: Cytoplasmic polyadenylation, Maternal gene, Mitogen-activated protein kinase (MAPK), M phase promoting factor (MPF) (J. Reprod. Dev. 57: [49][50][51][52][53][54][55][56] 2011) n mammals, fully grown follicular oocytes are arrested at the G2 stage, which is called the germinal vesicle (GV) stage of development. The oocytes resume meiosis in response to specific signals, usually hormones, or after being released from their follicular environment. Following germinal vesicle breakdown (GVBD), the chromatin is condensed, the metaphase I (MI) spindle is organized and oocytes extrude the first polar body upon completion of meiosis I. Subsequently, oocytes develop to metaphase of meiosis II (MII) and maintain the second meiotic arrest until fertilization [1][2][3]. Protein phosphorylation and dephosphorylation play pivotal roles in the oocyte meiotic cell cycle. Maturation/M-phase promoting factor (MPF) and mitogen-activated protein kinase (MAPK) are the principal regulatory systems that drive meiotic progression of oocytes. MAPK p42 and p44, also known as extracellular signal-regulated kinase 1 and 2 (ERK1 and ERK2), are serine/threonine kinases that can be phosphorylated and activated by MAPK kinase (MEK) [1,4]. MPF is a complex composed of a catalytic subunit, p34/cdc2, with serine/threon...