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.

Effects of Novel Probiotics in a Murine Model of Irritable Bowel Syndrome.

Background/Aims: Dysbiosis is an important factor in the pathogenesis of irritable bowel syndrome (IBS). Several studies have reported promising results using probiotics for the treatment of IBS. This study evaluated the efficacy of novel probiotics isolated from Kimchi, a Korean fermented food, and the feces of healthy Vietnamese people in a murine model of IBS. Methods: Lactobacillus paracasei DK121 was isolated from Kimchi, and L. salivarius V4 and L. plantarum V7 were isolated from the feces of healthy Vietnamese people residing in Korea. Forty rats were allocated to receive one of the study strains, a mixture of the strains, or the vehicle. After 5 days of administration, the rats were restrained in a cage to induce IBS. The effects of the probiotics on IBS were analyzed by evaluating the stool weights and stool consistency scores. Results: The primary outcome was analyzed upon the completion of a three-week experiment. The rats in the V7 group showed lower stool weights than those in the control group at week 2 (median: 1.10 [V7] vs. 2.35 [control], p=0.04, Mann-Whitney U-test) and week 3 (median: 1.10 [V7] vs. 2.80 [control], p=0.017). The rats in the DK121 (median: 2.00, p=0.007), V7 (median: 2.00, p=0.004), and mixture (median: 1.50, p=0.001) groups showed better stool consistency scores at week 2 than the control group (median: 3.00). Conclusions: The novel probiotics have beneficial effects on defecation in a murine model of IBS. Human studies confirming the efficacy are warranted.

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.

Transgenesis and nuclear transfer using porcine embryonic germ cells.

Embryonic germ (EG) cells are undifferentiated stem cells isolated from cultured primordial germ cells (PGC). Porcine EG cell lines with capacities of both in vitro and in vivo differentiation have been established. Because EG cells can be cultured indefinitely in an undifferentiated state, they may be more suitable for nuclear donor cells in nuclear transfer (NT) than somatic cells that have limited lifespan in primary culture. Use of EG cells could be particularly advantageous to provide an inexhaustible source of transgenic cells for NT. In this study the efficiencies of transgenesis and NT using porcine fetal fibroblasts and EG cells were compared. The rate of development to the blastocyst stage was significantly higher in EG cell NT than somatic cell NT (94 of 518, 18.2% vs. 72 of 501, 14.4%). To investigate if EG cells can be used for transgenesis in pigs, green fluorescent protein (GFP) gene was introduced into porcine EG cells. Nuclear transfer embryos using transfected EG cells gave rise to blastocysts (29 of 137, 21.2%) expressing GFP based on observation under fluorescence microscope. The results obtained from the present study suggest that EG cell NT may have advantages over somatic cell NT, and transgenic pigs may be produced using EG cells.

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.