Soon Young Heo

Resurrection of an alpha-1,3-galactosyltransferase gene-targeted miniature pig by recloning using postmortem ear skin fibroblasts

Animals with a targeted disruption of genes can be produced by somatic cell nuclear transfer (SCNT). However, difficulties in clonal selection of somatic cells with a targeted mutation often result in heterogeneous nuclear donor cells, including gene-targeted and non-targeted cells, and impose a risk of producing undesired wildtype cloned animals after SCNT. In addition, the efficiency of cloning by SCNT has remained extremely low. Most cloned embryos die in utero, and the few that develop to term show a high incidence of postnatal death and abnormalities. In the present study, resurrection of an alpha-1,3-galactosyltransferase (αGT) gene-targeted miniature pig by recloning using postmortem ear skin fibroblasts was attempted. Three cloned piglets were produced from the first round of SCNT, including one stillborn and two who died immediately after birth due to respiratory distress syndrome and cardiac dysfunction. Among the three piglets, two were confirmed to be αGT gene-targeted. Fibroblasts derived from postmortem ear skin biopsies were used as nuclear donor cells for the second round of SCNT, and a piglet was produced. As expected, PCR and Southern analyses confirmed that the piglet produced from recloning was αGT gene-targeted. Currently, the piglet is fourteen months of age, and no overt health problems have been observed. Results from the present study demonstrate that loss of an invaluable animal, such as a gene-targeted miniature pig, may be rescued by recloning, with assurance of the desired genetic modification.

Production of human CD59-transgenic pigs by embryonic germ cell nuclear transfer.

This study was performed to produce transgenic pigs expressing the human complement regulatory protein CD59 (hCD59) using the nuclear transfer (NT) of embryonic germ (EG) cells, which are undifferentiated stem cells derived from primordial germ cells. Because EG cells can be cultured indefinitely in an undifferentiated state, they may provide an inexhaustible source of nuclear donor cells for NT to produce transgenic pigs. A total of 1980 NT embryos derived from hCD59-transgenic EG cells were transferred to ten recipients, resulting in the birth of fifteen piglets from three pregnancies. Among these offspring, ten were alive without overt health problems. Based on PCR analysis, all fifteen piglets were confirmed as hCD59 transgenic. The expression of the hCD59 transgene in the ten living piglets was verified by RT-PCR. Western analysis showed the expression of the hCD59 protein in four of the ten RT-PCR-positive piglets. These results demonstrate that hCD59-transgenic pigs could effectively be produced by EG cell NT and that such transgenic pigs may be used as organ donors in pig-to-human xenotransplantation.

Targeted disruption of Ataxia-telangiectasia mutated gene in miniature pigs by somatic cell nuclear transfer

Ataxia telangiectasia (A-T) is a recessive autosomal disorder associated with pleiotropic phenotypes, including progressive cerebellar degeneration, gonad atrophy, and growth retardation. Even though A-T is known to be caused by the mutations in the Ataxia telangiectasia mutated (ATM) gene, the correlation between abnormal cellular physiology caused by ATM mutations and the multiple symptoms of A-T disease has not been clearly determined. None of the existing ATM mouse models properly reflects the extent to which neurological degeneration occurs in human. In an attempt to provide a large animal model for A-T, we produced gene-targeted pigs with mutations in the ATM gene by somatic cell nuclear transfer. The disrupted allele in the ATM gene of cloned piglets was confirmed via PCR and Southern blot analysis. The ATM gene-targeted pigs generated in the present study may provide an alternative to the current mouse model for the study of mechanisms underlying A-T disorder and for the development of new therapies.

Cytolytic assessment of hyperacute rejection and production of nuclear transfer embryos using hCD46-transgenic porcine embryonic germ cells

Human complement regulatory protein hCD46 may reduce the hyperacute rejection (HAR) in pig-to-human xenotransplantation. In this study, an hCD46 gene was introduced into porcine embryonic germ (EG) cells. Treatment of human serum did not affect the survival of hCD46-transgenic EG cells, whereas the treatment significantly reduced the survival of non-transgenic EG cells (p < 0.01). The transgenic EG cells presumably capable of alleviating HAR were transferred into enucleated oocytes. Among 235 reconstituted oocytes, 35 (14.9%) developed to the blastocyst stage. Analysis of individual embryos indicated that 80.0% (28/35) of embryos contained the transgene hCD46. The result of the present study demonstrates resistance of hCD46-transgenic EG cells against HAR, and the usefulness of the transgenic approach may be predicted by this cytolytic assessment prior to actual production of transgenic pigs. Subsequently performed EG cell nuclear transfer gave rise to hCD46-transgenic embryos. Further study on the transfer of these embryos to recipients may produce hCD46-transgenic pigs.

Gene targeting in mouse embryos mediated by RecA and modified single-stranded oligonucleotides

Gene targeting is a precise manipulation of endogenous gene by introduction of exogenous DNA and has contributed greatly to the elucidation of gene functions. Conventional gene targeting has been achieved through a use of embryonic stem cells. However, such procedure is often long, tedious, and expensive. This study was carried out to develop a simple procedure of gene targeting using E. coli recombinase A (RecA) and modified single-stranded oligonucleotides. The new procedure was attempted to modify X-linked hypoxanthine phosphoribosyltransferase (HPRT) gene in mouse embryos. The single-stranded oligonucleotide to target an exon 3 of HPRT was 74 bases in length including phosphorothioate linkages at each terminus to be resistant against exonucleases when introduced into zygotes. The oligonucleotide sequence was homologous to the target gene except a single nucleotide that induces a mismatch between an introduced oligonucleotide and endogenous HPRT gene. Endogenous repairing of such mismatch would give rise to the conversion of TAT to TAG stop codon thereby losing the function of the target gene. Before an introduction into zygotes, single-stranded oligonucleotides were bound to RecA to enhance the homologous recombination. The RecA–oligonucleotide complex was microinjected into the pronucleus of zygote. Individual microinjected embryos developed to the blastocyst stage were analyzed for the expected nucleotide conversion using polymerase chain reaction (PCR) and subsequent sequencing. The conversion of TAT to TAG stop codon was detected in three embryos among 48 tested blastocysts (6.25% in frequency). The result suggests that the gene targeting was feasible by relatively easier and direct method.

In vitro development of nuclear transfer embryos derived from porcine embryonic germ cells and their descendent neural precursor cells.

Undifferentiated stem cells may support a greater development of cloned embryos compared with differentiated cell types due to their ease of reprogramming during the nuclear transfer (NT) process. Hence, stem cells may be more suitable as nuclear donor cells for NT procedures than are somatic cells. Embryonic germ (EG) cells are undifferentiated stem cells that are isolated from cultured primordial germ cells (PGC) and can differentiate into several cell types. In this study, the in vitro development of NT embryos using porcine EG cells and their derivative neural precursor (NP) cells was investigated, thus eliminating any variation in genetic differences. The rates of fusion did not differ between NT embryos from EG and NP cells; however, the rate of cleavage in NT embryos derived from EG cells was significantly higher (p < 0.05) than that from NP cells (141/247 [57.1%] vs. 105/228 [46.1%]). Similarly, the rate of blastocyst development was significantly higher (P < 0.05) in NT using EG cells than the rate using NP cells (43/247 [17.4%] vs. 18/228 [7.9%]). The results obtained from the present study in pigs demonstrate a reduced capability for nuclear donor cells to be reprogrammed following the differentiation of porcine EG cells. Undifferentiated EG cells may be more amenable to reprogramming after reconstruction compared with differentiated somatic cells.

199 EFFECTS OF REPROGRAMMING-CONDITIONED MEDIUM ON ULTRAVIOLET RAY A–DAMAGED HUMAN DERMAL FIBROBLASTS

Ultraviolet ray A (UVA) is an electromagnetic light with a long wavelength from the sun. The penetration of UVA deep into the human dermis causes changes in cells, such as DNA fragmentation, apoptosis, and senescence, eventually leading a decline of proliferation and wound-healing ability. These changes induced by UVA exposure are similar to those seen in the process of stem cell differentiation. We postulated that the condition that reverses cellular differentiation may alleviate the UVA-induced damage in skin cells. Human dermal fibroblasts (HDF) could be reprogrammed to induced pluripotent stem cells (iPSC). Conditioned medium (CM) was prepared during the process of iPSC reprogramming (referred to as Repro-CM). The UVA-irradiated HDF were cultured in Repro-CM for 24h. In comparison with CM prepared from the culture of normal HDF and iPSC (referred to as HDF-CM and iPSC-CM, respectively), effects of Repro-CM on UVA-irradiated cells were investigated. Viability, wound-healing ability, apoptosis, and senescence of HDF were analysed by WST-1 assay, scratch assay, Annexin V assay, and senescence-associated β-galactosidase assay, respectively. Upon recovering from the UVA-induced damage, viability and wound-healing ability of HDF were significantly different (P<0.05) among the treatments in the order of Repro-, HDF-, and iPSC-CM. In the same context, apoptosis and senescence were significantly different (P<0.05) in the order of iPSC-, HDF-, and Repro-CM. Interestingly, iPSC-CM did not substantially ameliorate UVA-induced damage, suggesting that the conditions optimized to pluripotent stem cells may not be suitable for the recovery from damage in terminally differentiated cells, such as fibroblasts. The RNA-seq analysis was performed to assess the genome-wide transcriptional profile in the process of recovery. Repro- and HDF-CM were categorized more closely than iPSC-CM in hierarchical cluster analysis. In comparison with iPSC-CM, the up-regulated genes by Repro-CM treatment were related to regulation of cell proliferation and cell metabolism, whereas down-regulated genes were related to antiapoptosis and response to stimulation of chemical and organic substances. Overall, providing an environment of reprogramming, as shown by Repro-CM in the present study, may assist recovery of HDF from UVA-induced damage. The results of the study may be applicable in developing pharmaceuticals to treat aging and wrinkling of the skin caused by UVA irradiation.

Alpha-1,3-galactosyltransferase-deficient miniature pigs produced by serial cloning using neonatal skin fibroblasts with loss of heterozygosity.

Objective Production of alpha-1,3-galactosyltransferase (αGT)-deficient pigs is essential to overcome xenograft rejection in pig-to-human xenotransplantation. However, the production of such pigs requires a great deal of cost, time, and labor. Heterozygous αGT knockout pigs should be bred at least for two generations to ultimately obtain homozygote progenies. The present study was conducted to produce αGT-deficient miniature pigs in much reduced time using mitotic recombination in neonatal ear skin fibroblasts. Methods Miniature pig fibroblasts were transfected with αGT gene-targeting vector. Resulting gene-targeted fibroblasts were used for nuclear transfer (NT) to produce heterozygous αGT gene-targeted piglets. Fibroblasts isolated from ear skin biopsies of these piglets were cultured for 6 to 8 passages to induce loss of heterozygosity (LOH) and treated with biotin-conjugated IB4 that binds to galactose-α-1,3-galactose, an epitope produced by αGT. Using magnetic activated cell sorting, cells with monoallelic disruption of αGT were removed. Remaining cells with LOH carrying biallelic disruption of αGT were used for the second round NT to produce homozygous αGT gene-targeted piglets. Results Monoallelic mutation of αGT gene was confirmed by polymerase chain reaction in fibroblasts. Using these cells as nuclear donors, three heterozygous αGT gene-targeted piglets were produced by NT. Fibroblasts were collected from ear skin biopsies of these piglets, and homozygosity was induced by LOH. The second round NT using these fibroblasts resulted in production of three homozygous αGT knockout piglets. Conclusion The present study demonstrates that the time required for the production of αGT-deficient miniature pigs could be reduced significantly by postnatal skin biopsies and subsequent selection of mitotic recombinants. Such procedure may be beneficial for the production of homozygote knockout animals, especially in species, such as pigs, that require a substantial length of time for breeding.

35 In vitro development of nuclear transfer embryos derived from porcine embryonic germ cells and their descendent neural precursor cells

Due to an ease of reprogramming in the process of nuclear transfer (NT), undifferentiated stem cells may support greater development of cloned embryos compared with differentiated cell types. Hence, stem cells may be more suitable as nuclear donor cells for NT procedures than somatic cells. In pigs, comparative studies of NT using stem cells and their differentiated descendant cells have not yet been reported. Embryonic germ (EG) cells are undifferentiated stem cells isolated from cultured primordial germ cells (PGC) and can differentiate into several cell types. In this study, in vitro development of NT embryos using porcine EG cells and their derivative neural precursor (NP) cells was investigated. Porcine EG cells were isolated from cultured PGC collected from embryos on Day 23 of gestation (Shim et al. 1997 Biol. Reprod. 57, 1089–1095). Neural precursor cells, based on morphology of neural rosettes or neurospheres, were differentiated from porcine EG cells by sequential culture of the cells on gelatinized dishes in N2B27 medium with and without 10 µm retinoic acid for 7 days each. Since porcine EG cells and NP cells differentiated from the same EG cells were used in the present study, the genetic makeup of nuclear donor cells was identical, eliminating any genetic variation between undifferentiated and differentiated cells in the comparison. The efficiencies of NT using the two different cell populations were compared. The rates of fusion were not different between NT embryos from EG and NP cells. However, the rate of cleavage in NT embryos from EG cells was significantly higher (P < 0.05) than that from NP cells (141/247, 57.1% v. 105/228, 46.1%). Similarly, the rate of blastocyst development was significantly higher (P < 0.05) in NT using EG cells than that using NP cells (43/247, 17.4% v. 18/228, 7.9%). The effects on NT efficiency using either stem cells or their differentiated progeny is still controversial. The rates of blastocyst development using somatic stem cells were either reduced in mice (Sung et al. 2006 Nat. Genet. 38, 1323–1328) or remained the same in deer (Berg et al. 2007 Biol. Reprod. 77, 384–394) and cattle (Green et al. 2007 Biol. Reprod. 77, 395–406). However, the results obtained from the present study in pigs demonstrate a reduced capability for nuclear donor cells to be reprogrammed following the differentiation of porcine EG cells. This suggests that the effect on NT efficiency by using stem cells and their differentiated progeny may vary depending upon the species and cell types involved.

23 HYPOMETHYLATION OF DNA IN NUCLEAR TRANSFER EMBRYOS FROM PORCINE EMBRYONIC GERM CELLS

Epigenetic modification including genome-wide DNA demethylation is essential for normal embryonic development. Insufficient demethylation of the somatic cell genome may cause various anomalies and prenatal loss in the development of nuclear transfer embryos. Species-specific differences in the epigenetic status of cloned donor genomes have been reported. A level of DNA methylation in porcine somatic cell nuclear transfer (SCNT) embryos was similar to that of normally fertilized embryos, but hypermethylation of DNA in bovine SCNT embryos was commonly observed (Kang et al. 2001 J. Biol. Chem. 276, 39 980-39 984). Even in the same species, the source of the nuclear donor often affects later development of nuclear transfer embryos. In this study, appropriateness of porcine embryonic germ (EG) cells as karyoplasts for nuclear transfer with respect to epigenetic modification was investigated. These cells follow the methylation status of the primordial germ cells from which they originated, so they may contain a less methylated genome than somatic cells. The rates of blastocyst development were similar among embryos from EG cell nuclear transfer (EGCNT), SCNT, and intracytoplasmic sperm injection (ICSI) (16/62, 25.8% vs. 56/274, 20.4% vs. 16/74, 21.6%, respectively). Genomic DNA samples from EG cells (n = 3), fetal fibroblasts (n = 4), and blastocysts from EGCNT (n = 8), SCNT (n = 14), and ICSI (n = 6) were isolated and treated with sodium bisulfite. The satellite region (GenBank Z75640) that involves 9 selected CpG sites was amplified by PCR, and the rates of DNA methylation in each site were measured by pyrosequencing technique (Biotage AB, Uppsala, Sweden). The average methylation degrees of CpG sites in EG cells, fetal fibroblasts, and blastocysts from EGCNT, SCNT, and ICSI were 17.9, 37.7, 4.1, 9.8, and 8.9%, respectively. The genome of porcine EG cells was less methylated than that of somatic cells (P < 0.05), and DNA demethylation occurred in embryos from both EGCNT (P < 0.05) and SCNT (P < 0.01). However, the degree of DNA methylation in EGCNT embryos was approximately one-half that of SCNT (P < 0.01) and ICSI (P < 0.05) embryos; in SCNT and ICSI embryos, the genome was demethylated to the same degree. The present study demonstrated that porcine EG cell nuclear transfer results in hypomethylation of DNA in cloned embryos, yet leading to normal pre-implantation development. However, it would be interesting to further investigate whether such modification affects long-term survival of cloned embryos.