Y. W. Jeong

Production of Nuclear Transfer-Derived Piglets Using Porcine Fetal Fibroblasts Transfected with the Enhanced Green Fluorescent Protein

Abstract A system for somatic cell nuclear transfer (SCNT) was developed and led to the successful production of GFP-transfected piglets. In experiment 1, two groups of SCNT couplets reconstructed with porcine fetal fibroblasts (PFF) and enucleated sow (S) or gilt oocytes (G): 1) received a simultaneous electrical fusion/activation (S-EFA or G-EFA groups), or 2) were electrically fused followed by activation with ionomycin (S-EFIA or G-EFIA groups), or 3) were subjected to electrical fusion and subsequent activation by ionomycin, followed by 6-dimethylaminopurine treatment (S-EFIAD or G-EFIAD groups). The frequency of blastocyst formation was significantly higher in S-EFA (26%) compared with that observed in the other experimental groups (P < 0.05), but not with S-EFIA (23%). Sow oocytes yielded significantly higher cleavage frequencies (68%–69%) and total cell numbers of blastocysts when compared with gilt oocytes, regardless of fusion/activation methods (P < 0.05). However, the ratio of inner cell mass (ICM)/total cells in G-EFA and S-EFA was significantly lower than in the other groups (P < 0.05). In experiment 2, SCNT couplets reconstructed with PFF cultured in the presence or absence of serum and enucleated sow oocytes were subjected to EFA. There were no effects of serum starvation on cell-cycle synchronization, developmental competence, total cell numbers, and ratio of ICM/total cells. In experiment 3, SCNT couplets reconstructed with PFF transfected with an enhanced green fluorescence protein (EGFP) gene using FuGENE-6 and enucleated sow oocytes were subjected to EFA and cultured for 7 days. Expression frequencies of GFP gene during development were 100%, 78%, 72%, 71%, and 70% in fused, two-cell, four to eight cells, morulae, and blastocysts, respectively. In experiment 4, SCNT embryos derived from different recipient cytoplasts (sows or gilts) and donor karyoplasts (PFF or GFP-transfected) were subjected to EFA and transferred to the oviducts of surrogates. The pregnancy rates in SCNT embryos derived from sow oocytes (66%–69%) were higher than those with gilt oocytes (23%–27%) regardless of donor cell types. One live offspring from GFP-SCNT embryos and two from PFF-SCNT embryos were delivered. Microsatellite analysis confirmed that the clones were genetically identical to the donor cells and polymerase chain reaction (PCR) from genomic DNA of cloned piglets and subsequent southern blot analysis confirmed the integration of EGFP gene into chromosomes.

Anti-apoptotic effect of melatonin on preimplantation development of porcine parthenogenetic embryos

In the present study, we investigated the effect of melatonin on the preimplantation development of porcine parthenogenetic and somatic cell nuclear transfer (SCNT) embryos. Parthenogenetic embryos were cultured in mNCSU‐23 supplemented with various concentrations of melatonin for 7 days. The results revealed that 100 pM was the optimal concentration, which resulted in significantly increased cleavage and blastocyst formation rates. Additionally, 100 pM melatonin provided the highest increase in total cell number of blastocysts. Therefore, the subsequent experiments were performed with 100 pM melatonin. ROS level in 2–8 cell stage embryos in the presence or absence of melatonin was evaluated. Embryos cultured with melatonin showed significantly decreased ROS. Blastocysts cultured with melatonin for 7 days were analyzed by the TUNEL assay. It was observed that melatonin not only increased (Pu2009<u20090.05) the total cell number but also decreased (Pu2009<u20090.05) the rate of apoptotic nuclei. Blastocysts cultured with melatonin were assessed for the expression of apoptosis‐related genes Bcl‐xl and Bax, and of pluripotency marker gene Oct‐4 by real‐time quantitative PCR. Analysis of data showed that the expression of Bcl‐xl was higher (1.7‐fold) compared to the control while the expression of Bax was significantly decreased relative to the control (0.7‐fold) (Pu2009<u20090.05). Moreover, the expression of Oct‐4 was 1.7‐fold higher than the control. These results indicated that melatonin had beneficial effects on the development of porcine parthenogenetic embryos. Based on the findings of parthenogenetic embryos, we investigated the effect of melatonin on the development of porcine SCNT embryos. The results also demonstrated increased cleavage and blastocyst formation rates, and the total cell numbers in blastocysts were significantly higher when the embryos were cultured with melatonin. Therefore, these data suggested that melatonin may have important implications for improving porcine preimplantation SCNT embryo development. Mol. Reprod. Dev. 75: 1127–1135, 2008.

Production of Multiple Transgenic Yucatan Miniature Pigs Expressing Human Complement Regulatory Factors, Human CD55, CD59, and H-Transferase Genes

The present study was conducted to generate transgenic pigs coexpressing human CD55, CD59, and H-transferase (HT) using an IRES-mediated polycistronic vector. The study focused on hyperacute rejection (HAR) when considering clinical xenotransplantation as an alternative source for human organ transplants. In total, 35 transgenic cloned piglets were produced by somatic cell nuclear transfer (SCNT) and were confirmed for genomic integration of the transgenes from umbilical cord samples by PCR analysis. Eighteen swine umbilical vein endothelial cells (SUVEC) were isolated from umbilical cord veins freshly obtained from the piglets. We observed a higher expression of transgenes in the transgenic SUVEC (Tg SUVEC) compared with the human umbilical vein endothelial cells (HUVEC). Among these genes, HT and hCD59 were expressed at a higher level in the tested Tg organs compared with non-Tg control organs, but there was no difference in hCD55 expression between them. The transgenes in various organs of the Tg clones revealed organ-specific and spatial expression patterns. Using from 0 to 50% human serum solutions, we performed human complement-mediated cytolysis assays. The results showed that, overall, the Tg SUVEC tested had greater survival rates than did the non-Tg SUVEC, and the Tg SUVEC with higher HT expression levels tended to have more down-regulated α-Gal epitope expression, resulting in greater protection against cytotoxicity. By contrast, several Tg SUVEC with low CD55 expression exhibited a decreased resistance response to cytolysis. These results indicated that the levels of HT expression were inversely correlated with the levels of α-Gal epitope expression and that the combined expression of hCD55, hCD59, and HT proteins in SUVECs markedly enhances a protective response to human serum-mediated cytolysis. Taken together, these results suggest that combining a polycistronic vector system with SCNT methods provides a fast and efficient alternative for the generation of transgenic large animals with multiple genetic modifications.

X-linked gene transcription patterns in female and male in vivo, in vitro and cloned porcine individual blastocysts.

To determine the presence of sexual dimorphic transcription and how in vitro culture environments influence X-linked gene transcription patterns in preimplantation embryos, we analyzed mRNA expression levels in in vivo-derived, in vitro-fertilized (IVF), and cloned porcine blastocysts. Our results clearly show that sex-biased expression occurred between female and male in vivo blastocysts in X-linked genes. The expression levels of XIST, G6PD, HPRT1, PGK1, and BEX1 were significantly higher in female than in male blastocysts, but ZXDA displayed higher levels in male than in female blastocysts. Although we found aberrant expression patterns for several genes in IVF and cloned blastocysts, similar sex-biased expression patterns (on average) were observed between the sexes. The transcript levels of BEX1 and XIST were upregulated and PGK1 was downregulated in both IVF and cloned blastocysts compared with in vivo counterparts. Moreover, a remarkable degree of expression heterogeneity was observed among individual cloned embryos (the level of heterogeneity was similar in both sexes) but only a small proportion of female IVF embryos exhibited variability, indicating that this phenomenon may be primarily caused by faulty reprogramming by the somatic cell nuclear transfer (SCNT) process rather than in vitro conditions. Aberrant expression patterns in cloned embryos of both sexes were not ameliorated by treatment with Scriptaid as a potent HDACi, although the blastocyst rate increased remarkably after this treatment. Taken together, these results indicate that female and male porcine blastocysts produced in vivo and in vitro transcriptional sexual dimorphisms in the selected X-linked genes and compensation of X-linked gene dosage may not occur at the blastocyst stage. Moreover, altered X-linked gene expression frequently occurred in porcine IVF and cloned embryos, indicating that X-linked gene regulation is susceptible to in vitro culture and the SCNT process, which may eventually lead to problems with embryonic or placental defects.

Morphological abnormalities, impaired fetal development and decrease in myostatin expression following somatic cell nuclear transfer in dogs

Several mammals, including dogs, have been successfully cloned using somatic cell nuclear transfer (SCNT), but the efficiency of generating normal, live offspring is relatively low. Although the high failure rate has been attributed to incomplete reprogramming of the somatic nuclei during the cloning process, the exact cause is not fully known. To elucidate the cause of death in cloned offspring, 12 deceased offspring cloned by SCNT were necropsied. The clones were either stillborn just prior to delivery or died with dyspnea shortly after birth. On gross examination, defects in the anterior abdominal wall and increased heart and liver sizes were found. Notably, a significant increase in muscle mass and macroglossia lesions were observed in deceased SCNT‐cloned dogs. Interestingly, the expression of myostatin, a negative regulator of muscle growth during embryogenesis, was down‐regulated at the mRNA level in tongues and skeletal muscles of SCNT‐cloned dogs compared with a normal dog. Results of the present study suggest that decreased expression of myostatin in SCNT‐cloned dogs may be involved in morphological abnormalities such as increased muscle mass and macroglossia, which may contribute to impaired fetal development and poor survival rates. Mol. Reprod. Dev. 78:337–346, 2011.

Analysis of imprinted IGF2/H19 gene methylation and expression in normal fertilized and parthenogenetic embryonic stem cells of pigs

To determine whether the genomic imprinting can be maintained during the process of embryonic stem (ES) cell derivation from pig blastocysts, mRNA and DNA methylation at the IGF2/H19 imprinting control region in putative ES cells derived from in vitro fertilized (IVF) and parthenogenetic (PG) embryos were investigated. In the present study, one IVF- and three PG ES-like cell lines were established and analyzed for cellular characteristics such as pluripotent marker expression and differentiation capacity. The results showed that these putative ES cells derived from pig blastocysts fulfilled the general stemness criteria. The expression of the H19 gene was significantly greater in PG blastocysts than IVF blastocysts, but there were greater amounts of IGF2 in IVF than PG blastocysts. Of these putative ES cell lines, one PG line had less H19 gene expression than a IVF ES cell line while the other two PG lines had much greater expression of the H19 gene than the IVF line. In contrast, the IGF2 gene was upregulated in the same PG cell line relative to the other two PG cell lines and transcript abundance was similar to IVF ES-like cells. Despite the variable amounts of mRNA among the PG cell lines, the IGF2/H19 gene had a differentially methylated region (DMR) 3 was typically un-methylated in all PG cells, and hemi-methylated in the IVF cells. These findings indicated that the mRNA of H19 and IGF2 genes is susceptible to in vitro environments during the process of ES cell derivation from blastocysts but DNA methylation status at this region was well maintained. These altered gene expressions may not be associated with the methylation of the imprinting control region at this locus. Therefore, with their uni-parental genotype, the pluripotent differentiation potentials of PG ES cells could be a valuable tool for understanding genomic imprinting in embryonic development.

56 Production of transgenic recloned miniature pigs expressing human decay accelerating factor.

This study was performed to produce transgenic recloned miniature pigs expressing human decay-accelerating factor (hDAF) to overcome the hyperacute rejection of pig-to-human xenotransplantation. The expression vector, named hDAF-Neo, was constructed by subcloning the amplified 2.2 kb hDAF cDNA fragment into the NheI and NotI site of the pEGFP-N1 (Clontech, Palo Alto, CA, USA) vector containing the chicken beta-actin promoter and rabbit globin poly A without a EGFP fragment. Day 30 fetal fibroblast cells (GN0) were used for producing the newborn cloned pigs (GN1). GN1 fibroblasts cultured from the newborn cloned pig derived from GN0 were used for transfection of the hDAF-Neo plasmid using FuGENE-6® (Roche Diagnostics, Indianapolis, IN, USA). Transfected fibroblast cell colonies were selected with neomycin. All data were analyzed by one-way ANOVA and the protected least significant difference (LSD) test using general linear models in a statistical analysis system (SAS Institute, Inc., Cary, NC, USA) program to determine differences among experimental groups. Significant difference among the treatment groups was determined when the P value was less than 0.05. There was no significantly different development rate between GN0 and GN1 (12.8 vs. 13.0%) and between GN1-hDAF and GN2 (9.3 vs. 8.2%). Transfected recloned embryos (GN1-hDAF) had no significantly different blastocyst formation compared to normal (GN1). After embryo transfer, we obtained two transgenic recloned pigs. hDAF gene integration in transgenic recloned piglets was confirmed by polymerase chain reaction. Cultured fibroblasts of transgenic recloned pigs showed 1.0-2.3 times higher levels of hDAF protein than did human umbilical vein endothelial cells. The levels of C3 deposition on cultured fibroblast cells after incubation with 10% human serum were decreased in transgenic recloned pigs compared to normal miniature pigs. In conclusion, the recloning procedure can be used to produce multiple genetically modified cloned pigs without any severe epigenetic abnormality. This study was supported by grants from the Korean MOST (Top Scientist Fellowship) and MAF (Biogreen 21 #20050301-034-443-026-01-00).