Abstract
Abnormalities in gene expression that negatively affect embryonic development are frequently observed in cloned embryos generated by somatic cell nuclear transfer (SCNT). In the present study, we successfully produced a cell-penetrating peptide (CPP)-conjugated with coactivator-associated arginine methyltransferase 1 (CARM1) protein from mammalian cells and confirmed introduction into donor somatic cells and cloned 8-cell embryos within 3 hours after addition to culture medium. In addition, H3R17 dimethylation and embryonic development up to the blastocyst stage were increased in the group treated with exogenous CPP-CARM1 protein compared with the untreated group (control). Interestingly, the number of total cells and trophectoderm in blastocysts as well as implantation rate were significantly increased in the CPP-CARM1 protein-treated group. However, the cell number of inner cell mass (ICM) was not changed compared with the control group; similarly, expression of pluripotency-related genes Oct4 and Nanog (ICM markers) was not significantly different between groups. On the other hand, expression of the implantation-related gene Cdx2 (trophectoderm marker) was transiently increased after treatment with CPP-CARM1 protein. On the basis of these results, we conclude that supplementation with exogenous CPP-CARM1 protein improves embryonic development of cloned embryos through regulation of histone methylation and gene expression. In addition, our results suggest that CPP-CARM1 protein may be a useful tool for strengthening implantation of mammalian embryos.
Highlights
Somatic cell nuclear transfer (SCNT) is the process by which the cytoplasm of a recipient oocyte reprograms a nucleus from a differentiated somatic donor cell, resulting in the production of cloned embryos with the genetic information of the donor cell
It was recently reported that induced pluripotent cells could be generated by introducing transcription factors fused with a cell-penetrating peptide (CPP)[21,22]
To enable efficient delivery of recombinant proteins, we fused a CPP (KRK) sequence to the C-terminus of each protein; purification was facilitated by incorporating a FLAG- and His6-tag at the C-terminal end of DsRed2- and coactivator-associated arginine methyltransferase 1 (CARM1)-constructs (Fig. 1A)
Summary
Somatic cell nuclear transfer (SCNT) is the process by which the cytoplasm of a recipient oocyte reprograms a nucleus from a differentiated somatic donor cell, resulting in the production of cloned embryos with the genetic information of the donor cell This technique was first described for the embryonic development of Xenopus in a report by Gurdon[1]. Poor quality of developmental patterns attributable to reduced cell number and altered gene expression were frequently observed in most cloned embryos compared with in vivo- and in vitro-fertilized embryos[5] To overcome this problem, researchers have sought to produce improved cloned embryos through supplementation of the culture medium with chemical reagents[6,7,8] and growth factors[9,10]. We provide the first demonstration that exogenous supplementation with a novel CPP-conjugated CARM1 improves the normally poor embryonic development of cloned mouse embryos through regulation of gene expression
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