Somatic cell nuclear transfer (SCNT) is a useful biotechnological tool for animal cloning. Until now, SCNT has been inefficient, especially in dog. It is believed that an embryo developmental block in SCNT embryos is cause of low production efficiency. However, no studies have been performed on canines for embryo developmental block. In this study, we attempted to evaluate the beneficial role of EDTA in canine parthenogenic (PA) embryos development to overcome embryo developmental block. The PA embryos were divided into 0.01 mM EDTA treated and non-treated groups. Embryo developmental efficiency was measured by activating chemically parthenote. After EDTA induction, PA embryos were evaluated for embryonic development, Reactive Oxygen Species (ROS) activity, mitochondrial integrity, ATP production and genomic activation. The EDTA treated PA embryos showed significantly higher survival rate and improved cavity formation compared to non-treated. Furthermore, cytoplasmic ROS level was mitigated and mitochondrial membrane potential was found significantly higher in EDTA treated group followed by higher ATP production. Moreover, major embryonic genomic activation specific markers/factors were also elevated in EDTA treated group. Conclusively, we elucidated that EDTA showed substantially positive effect to overcome embryo developmental block in canine.

Alzheimer's disease (AD) has caused by expression of amyloid precursor protein (APP), Tau and presenilin (PS) as known as plaques and tangle accumulation. AD transgenic porcine model is necessary for preclinical testing of therapeutic agent because of similar metabolic system between porcine and human. The objective of study was to generate AD transgenic pig by somatic cell nuclear transfer (SCNT) with multi-cistronic vector system. AD multi-cistronic vector was 6 well-known mutation on 3 AD related genes, hAPP (K670N/M671L, I716V, V717I), hTau (P301L) and hPS1 (M146V, L286P). Establishment of AD transgenic cell lines was used from Jeju black pig ear fibroblast cells (JB-PEFAD) with the AD multi-cistronic vector. The JB-PEFAD cell was confirmed on mRNA expression, protein synthesis of hAPP, hTau and hPS1 and identification of integration and karyotype. Although fusion rate was no difference in SCNT with JB-PEF AD (SCNTAD) embryos, cleavage and blastocyst formation rates were slightly lower than in SCNT with non-transgenic JB-PEF (SCNTnon-TG). Individual SCNTAD blastocysts were detected hAPP, hTau and hPS1 genomic integration which showed 93.2% (n=30) efficiency in genomic DNA (gDNA) level. It will give us a possibility to develop porcine animal model for AD study in the future.

This study investigated the developmental ability and gene expression of somatic cell nuclear transfer embryos using ear skin fibroblast cells derived from miniature pig. When miniature pig (m) and landrace pig (p) were used as donor cells, there were no differences in cleavage (79.2 vs. 78.2%) and blastocyst rates (27.4 vs. 29.7%). However, mNT blastocysts showed significantly higher apoptosis rate than that of pNT blastocysts (6.1 vs. 1.7%) (p<0.05). The number of nuclei in pNT blastosysts was significantly higher than that of mNT (35.8 vs. 29.3) (p<0.05). Blastocysts were analyzed using Realtime RT-PCR to determine the expression of Bax-, Bcl-xl, H19, IGF2, IGF2r and Xist. Bax- was higher in mNT blastocyst than pNT blastocyst (p<0.05). There was no difference in Bcl-xl between two NT groups. Bax-/Bcl-xl was, however, significantly higher in mNT blastocyst compared to pNT. The expression of imprinting genes were aberrant in blastocysts derived from NT compared to in vivo blastocysts. H19 and IGF2r were significantly lower in mNT blastocysts (p<0.05). The expression of IGF2 and Xist was similar in two NT groups. However, imprinting genes were expressed aberrantly in mNT compared to pNT blastocysts. The present results suggest that the NT between donor cells derived from miniature pig and recipient oocytes derived from crossbred pig might affect reprogramming of donor cell, resulting in high apoptosis and aberrant expression patterns of imprinting genes.