Development of a Noninvasive Monitoring System for Evaluation of Oct-3/4 Promoter Status in Miniature Pig Somatic Cell Nuclear Transfer Embryos

Abstract

Abstract. The present study was carried out to develop a noninvasive monitoring system for evaluation of Oct-3/4 promoter gene status in miniature pig somatic cell nuclear transfer (SCNT) embryos during in vitro development. Miniature pig fetal fibroblasts (MPFFs) were transfected with a gene construct consisting of two expression units, a mouse Oct-3/4 promoter-driven enhanced green fluorescent protein (EGFP) gene (EGFP expression only detected in Oct-3/4-expressing cells) and a neomycin resistance gene. After neomycin selection, MPFFs that did not express EGFP were fused with enucleated pig oocytes, cultured in vitro and assessed for EGFP expression. EGFP expression was detectable in all morulae (at 4-6 days of culture) and 50.0% of blastocysts (at 5-6 days of culture), whereas none of the 1-cell to 16-cell embryos at 1-5 days of culture expressed EGFP. On the other hand, EGFP expression was not maintained in all blastocysts at 7 days of culture. The reactivity with anti-Oct-3/4 antibodies also peaked from the morula to blastocyst stages at 5 days of culture. The results showed that reactivation of the Oct-3/4 promoter gene of donor nuclei occurs in the morula to blastocyst stages at 4-6 days after SCNT and that this noninvasive monitoring system using Oct-3/4 promoter-driven EGFP gene would be useful for evaluation of the reprogramming status of donor nuclei. Key words: Enhanced green fluorescent protein, In vitro development, Nuclear transfer, Oct-3/4 promoter, Reprogramming (J. Reprod. Dev. 55: [661][662][663][664][665][666][667][668][669] 2009) uccessful production of cloned animals using somatic cell nuclear transfer (SCNT) has been reported in several mammalian species [1][2][3][4][5][6][7][8][9][10][11][12]. However, the low cloning efficiency associated with development of SCNT embryos to offspring remains the major obstacle to widespread use of this technology in a number of animal science and biomedical applications. A method for evaluating the developmental ability of SCNT embryos before being transferred into recipient females should be explored to optimize the procedures of SCNT and overcome the low cloning efficiency. Evaluation of the in vitro developmental ability of an SCNT embryo to the blastocyst stage would be considered one of the methods for predicting its in vivo development, but it has been found that this does not often assure successful development to term of SCNT embryos [13]. After SCNT, the gene expression pattern in somatic cells is reprogrammed to mimic that in preimplantation embryos [14]. However, there is a difference in gene expression pattern between SCNT embryos and in vitro-fertilized embryos, suggesting that the low efficiency of SCNT is associated with incomplete reprogramming in the donor nuclei transferred into recipient oocytes [15]. Therefore, evaluation of the reprogramming level in the donor nuclei appears to be most essential for predicting the in vivo developmental ability of SCNT embryos.Oct-3/4 is a transcription factor of the Pit-Oct-Unc family [16,17], and...

Successful Suppression of Endogenous <i>&alpha;-1,3-Galactosyltransferase</i> Expression by RNA Interference in Pig Embryos Generated <i>In Vitro</i>

“…4C, lane 8) may be ascribed to failure to express their α-GalT mRNA at a level detectable by RT-PCR, as previously pointed out. This phenomenon may be related to the failure of proper reprogramming of donor nuclei after SCNT, since gene activation of embryonic and pluripotency-related genes is sometimes suppressed [43,44]. In our preliminary data, expression of α-GalT mRNA increased gradually with progression towards the blastocyst stage (unpublished results).…”

Beneficial Effects of Reversine on In Vitro Development of Miniature Pig Somatic Cell Nuclear Transfer Embryos

“…The resulting embryos exhibited EGFP expression at the morula to blastocyst stages. Immunohistochemical staining using anti-Oct-3/4 antibodies revealed that the EGFP expression pattern seen in these SCNT embryos was correlated with that of endogenous Oct-3/4 gene [16]. Therefore, this Oct-3/4-EGFP-based noninvasive monitoring system would be useful for evaluation of the reprogramming level of SCNT embryos in miniature pigs.…”

“…In addition, the Oct-3/4 expression pattern in the tissues of adult pigs is the same as that in adult mice; Oct-3/4 is expressed in PGCs, the ovary and the testis, but not in the heart, liver, lung, kidney, spleen and fibroblasts [15]. Based on this background, Oct-3/4 appears to be a good marker for evaluating the reprogramming level of SCNT embryos.In the previous studies [16,17], we produced SCNT embryos by transferring miniature pig fetal fibroblasts (MPFFs) transfected with a plasmid (termed pOEIN) that contains a mouse Oct-3/4 promoter-driven enhanced green fluorescent protein (EGFP) cDNA into enucleated pig oocytes. The resulting embryos exhibited EGFP expression at the morula to blastocyst stages.…”

“…A single pOEIN-MPFF-#4 cell was inserted into the perivitelline space of each enucleated oocyte using the same glass pipette. Membrane fusion of the cytoplast and donor cell was induced by applying a single direct-current pulse [16,17]. Following the fusion pulse, cell-oocyte complexes were cultured for 2 h in 100 μl of modified PZM-3 (mPZM-3) [21] until activation.…”

“…In the previous studies [16,17], we produced SCNT embryos by transferring miniature pig fetal fibroblasts (MPFFs) transfected with a plasmid (termed pOEIN) that contains a mouse Oct-3/4 promoter-driven enhanced green fluorescent protein (EGFP) cDNA into enucleated pig oocytes. The resulting embryos exhibited EGFP expression at the morula to blastocyst stages.…”