• Genomics & Informatics
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• v.8 no.4
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• pp.201-205
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• 2010

The H1 histone family, member N, testis-specific (H1FNT) is exclusively expressed in the testis, and had its possible role for sperm chromatin formation. The purpose of this study is to investigate any genetic association of H1FNT gene with male infertility, especially at the promoter region. We examined the promoter single nucleotide polymorphisms (SNP) of H1FNT gene which is located within transcription factor binding site for its association with male infertility. The statistical analysis showed that the -1129A>T polymorphism was present at a statistically significance in male infertility (p=0.0059 and 0.0349 for hetero and risk type, respectively). The dual-luciferase promoter assay was performed to examine the polymorphic effect of this promoter SNP by the cloning of promoter region (1700bp fragment) into pGL3-basic vector. In our plasmid based reporter system, there is no big difference between wild and risk type. In conclusion, H1FNT -1129A>T promoter SNP is statistically significant with male infertility, especially with subfertile (non-azoospermia) group. Further analysis of its functional polymorphic effect in vivo may provide the biological significance of testis-specific histone with spermatogenesis.

• Journal of Life Science
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• v.10 no.1
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• pp.40-44
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• 2000

The SNF2/SW ATPase/helicase family comprises proteins form a variety of species with in vivo functions, such as transcriptional regulation, maintenance of chromosome stability during mitosis, and various types of DNA repair. Here, we reported the characterization of h게2+gene which was iolated by PCR amplification using the conserved domain of SNF2 motifs. Sequence analysis of PCR product showed striking evolutionary conservation among the SNF2 family of proteins. Two transcripts of 6.7 and 3.4 Lb were detected by Northern blot analysis. furthermore, the intensities of these two bands were increased by ultraviolet(UV) irradiation. These results indicate that the hrp2+ is a novel member of the SNF2 family of proteins and is one of the UV-inducible genes in S. pombe. To determine the level of transcripts of hrp2+ gene during cellular growth, Northern blot analysis were performed. This result indicates that the level of hrp2+transcript reached its maximum before cells entered the exponential growth phase. This suggests that hrp2+ gene is experssed mainly at the early stage of cell growth.

• Journal of Embryo Transfer
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• v.10 no.3
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• pp.209-217
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• 1995

The present study was carried out to develop a cloning technology of mouse embryos by nuclear transplantation with electrofusion and to produce cloned offsprings by transfer of reconstituted embryos. A single nucleus from two- and eight-cell embryos was transplanted into the enucleated two-cell embryos by rnicromanipulation. The fusion of nucleus with recipient cytoplasm and the subsequent development of reconstituted embryos in vitro as well as in vivo to term were examined to determine the optimal electrofusion parameters for nuclear transplantation in mouse embryos. The successful enucleation of donor embryos was 84.9 and 83.3% in two- and eight-cell stage, respectively, and the successful injection of nucleus from two- and eight-cell donor embryos into the perivitelline space of enucleated two-cell embryos were 85.1 and 84.7%, respectively. No significant differences were found in enucleation or injection rate between the cell stages of donor embryos. When the blastomeres of intact two-cell mouse embryos were electrofused in 0.3 M mannitol medium(100 $\mu$sec., 3 pulses), the fusion rate was similarly 93.2, 92.2 and 92.0% in 1.0, 1.5 and 2.0 kV /crn, respectively, but in vitro development to blastocyst of the fused two-cell embryos was significantly(P<0.05) lower in 2.0 kV/cm (63.4%) than in 1.0 kV/cm (91.7%) or 1.5 kV/cm (82.4%). The development in vitro to eight-cell stage of the reconstituted embryos with nucleus from two-cell stage(45.5%) was significantly(P<0.05) higher than that from eight-cell stage blastomeres (16.7%). The number of blastomeres of the intact embryos at blastocyst stage was 50i0.6 and 55$\pm$2.4 in in vitro and in vivo cultured mouse embryos, respectively, but significantly(P<0.05) decreased to 35$\pm$0.7 in nuclear transplanted blastocyst embryos. The conception rate of mice following embryo transfer was 32.1% in the reconstituted two-cell embryos using two-cell donor nuclei, which was comparable to the fresh two-cell embryos(40.6%). However, the rate of development in vivo to term following embryo transfer of the reconstituted two-cell embryos using two-cell donor nuclei (23.5%) was significantly(P<0.05) lower compared with the percentage of two-cell fresh embryos(31.5%).

• Applied Biological Chemistry
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• v.42 no.4
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• pp.293-297
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• 1999

The pts operon, which encodes several factors in the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) of Escherichia coli, has multiple promoters which respond to different signals to facilitate quick adaptation to changes in growth conditions. The influence of an 1 kbp DNA region upstream of the pts P0 promoter on pts expression was studied in vitro by employing the DNA templates containing both P0 and P1 promoter with or without the 1 kbp upstream DNA region for in vitro transcription assay. The 1 kbp DNA region upstream of the pts P0 promoter, however, had no effect on pts transcription in vitro. The intracellular concentration of cAMP was measured when cells were grown in the presence of glucose, mannose, or mannitol. The transcription of P0 was increased maximally in the presence of glucose even though the concentration of cAMP in the condition was lowest while the transcription from the P1b was highest when cells were grown in the presence of mannose or mannitol even though the intracellular concentration of cAMP was lower than cells grown in the absence of the sugar. These results suggest the possibility of the existence of a glucose inducible repressor specific for the P0 promoter and a second repressor that is inducible by glucose, mannose and mannitol specific for the P1 promoter.

• Korean Journal of Poultry Science
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• v.48 no.3
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• pp.111-122
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• 2021

Recently, interleukin 34 (IL-34) was identified as the second functional ligand for macrophage colony-stimulating factor receptor (M-CSFR). IL-34 functions similarly to M-CSF through its binding to the M-CSFR. There is still insufficient information on IL-34 in chickens, which has until now been reported only through predicted sequences and not through experimental research. Thus, to confirm its expression and to determine its potent biological activity, several chicken lines and cell lines were used. Cloning of recombinant chicken IL-34 and M-CSF genes was performed to investigate their modulatory effects on proinflammatory cytokine expression in vitro. The expression levels of IL-34, M-CSF, and M-CSFR genes were upregulated in broiler chickens with leg dysfunction (cause unknown). However, IL-34 was downregulated in most pathogen-stimulated tissues. M-CSFR expression was enhanced by recombinant IL-34 and M-CSF proteins in vitro. IFN-γ expression was enhanced by recombinant IL-34, but not by M-CSF. However, IL-12 expression was not regulated in any of the treated cells, and IL-1β was decreased in all tissues. These results indicate that IL-34 and M-CSF have roles in both the classical and alternative macrophage activation pathways. Collectively, our findings demonstrate the expression of IL-34 in chickens for pathogenic trials, both in vitro and in vivo. Our results suggest that the IL-34 protein plays a role in both pro- and anti-inflammatory functions in macrophages. Therefore, further research is needed to determine the cytokines or chemokines that can be induced by IL-34 and to further elucidate the functions of IL-34 in the inflammatory pathway.

• Proceedings of the KSAR Conference
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• 2001.03a
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• pp.62-62
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• 2001

Recently, production of transgenic animal by nuclear transfer has been known as a useful method. The production of cloned offspring derived from nuclear transfer depends upon a variety of factors such as species, donor cells type and cell cycle, and source of recipient ova. Therefore, we attempted a different transgenic methods using follicular granulosa cells (GCs). In general, ovulated GCs undergoes lutenization and transformation in vitro which might defective effects on developmental potential. In order to avoid the GCs transformation in vitro culture system, we introduced a direct injection of retrovirus into the follicles and then collected them mechanically from ovaries of 6-8 week-old ICR mice. Retrovirus vector constructed with pLN $\beta$ EGFP was injected into the follicles. The follicles are cultured in $\alpha$ -MEM supplemented with human FSH, LH and ITS in Costar Transwell dish for 4 days. Survival rate of virus injected follicles was 52.1% (12/23) and expression rate of EGPP gene was 33.3% (4/12). In this study, we found GCs performed transgenesis in our culture system. In addition, the GCs in follicle may be developed in vivo like environment rather than in vitro environment. Thus, the use of GCs as donor cells may be useful in the nuclear transfer for cloning of genetic modification. Therefore, these results suggest that follicular GCs can be transfected by viral vector during folliculogenesis in vitro.

• Asian-Australasian Journal of Animal Sciences
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• v.16 no.7
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• pp.1071-1086
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• 2003

Reproductive biotechnologies continue to be developed for genetic improvement of both river and swamp buffalo. Although artificial insemination using frozen semen emerged some decades back, there are still considerable limitations. The major problem appears to be the lack of efficient methods for estrus detection and timely insemination. Controlled breeding experiments in the buffalo had been limited and similar to those applied in cattle. Studies on multiple ovulation and embryo transfer are essentially a replica of those in cattle, however with inherent problems such as lower number of primordial follicles on the buffalo ovary, poor fertility and seasonality of reproduction, lower population of antral follicles at all stages of the estrous cycle, poor endocrine status and a high incidence of deep atresia in ovarian follicles, the response in terms of transferable embryo recovery has remained low with 0.51 to 3.0 per donor and pregnancy rates between 15 to 30%. In vitro production of buffalo embryos is a valid alternative to recovery of embryos by superovulation. This aspect received considerable attention during the past decade, however the proportion of embryos that develops to the blastocyst stage is still around 25-30% and hence the in vitro culture procedures need substantial improvement. Embryo cryopreservation procedures for direct transfer post thaw need to be developed for bubaline embryos. Nuclear transfer and embryo cloning is a technique that has received attention in various species during recent years and can be of immense value in buffaloes as they have a low rate of embryo recoveries by both in vitro and in vivo procedures. Gender pre-selection, genome analysis, gene mapping and gene transfer are a few of the techniques that have been studied to a limited extent during recent years and are likely to be included in future studies on buffaloes. Very recently, reproductive biotechnologies have been applied to feral buffaloes as well, but the results obtained so far are modest. When fully exploited they can play an important role in the preservation of endangered species.

• Korean Journal of Veterinary Research
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• v.30 no.4
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• pp.355-360
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• 1990

Single nuclei from two-, four- and eight-cell mouse embryos were transplanted into enucleated two-cell embryos by micromanipulation and Sendai virus mediated fusion. The developmental potential of these reconstituted embryos in vitro and in vivo was examined. It was found that the single nuclei which were transplanted to enucleated two-cell embryos were not only able to develop to the blastocyst stage in vitro(two-cell nuclei, 76.5%; four-cell nuclei, 68.4%; eight-cell nuclei, 48.3%), but also able to develop to full term in vivo after transfer to recipient mice(two-cell nuclei, 37.1%; four-cell nuclei, 29.6%; eight-cell nuclei, 16.3%). Although the proportion of live young produced after transfer of nucler of nuclear transplant embryos which received eight-cell nuclei was significantly (p<0.05) reduced, it would be suggested that the overall efficiency in producing identical offspring is greater when eight-cell embryos were selected for nuclear donor than two- or four-cell embryos were selected.

• Reproductive and Developmental Biology
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• v.33 no.4
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• pp.195-202
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• 2009

Abnormal development and fetal loss during the post-implantation period are key concerns in the production of cloned animals by somatic cell nuclear transfer (SCNT). We hypothesized that the problems in cloned porcine offspring derived from SCNT are related to interactions between the conceptus and the endometrial environment. In the present study, we investigated expression patterns in the formation of placenta-related genes (Cdx2 and GATA6) in whole in vivo normal porcine embryos (from single cell to blastocyst) and each tissue of a normal fetus at Days 25, 35 and 55 by quantitative mRNA expression analysis using real-time PCR. The expression of Cdx2 and GATA6 mRNA increased to around the blastocyst stage. These genes were gradually decreased from the peri-implantation to post-implantation stage. Moreover, we examined the expression patterns of Cdx2 and GATA6 in Day 35 normal and SCNT cloned fetuses by the same methods. And, the level of Cdx2 and GATA6 gene expression in the extraembryonic tissue of SCNT was significantly higher than that of control tissues. From the present results, it can be postulated that the aberrant expression of Cdx2 and GATA6 genes in the endometrial and extraembryonic tissues at pre- and peri-implantation stages may be closely related to the lower efficiency of animal cloning.

• Microbiology and Biotechnology Letters
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• v.35 no.1
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• pp.36-40
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• 2007

Redox signaling is one of way to regulate growth and death of cell in response to change of redox of proteins. To search whether translation is regulated by redox, we attempted in vitro translation assay under condition with or without DTT. Interestingly in vitro translation activity was increased up to 40% In the presence of dithiothreitol (DTT). Then we checked whether this positive effect by DTT was further accelerated by addition of thioredoxin (Trx). When a Trx purified from Saccharomyces cerevisiae was added to the in vitro translation extract, we observed a dose-dependent increase in translational activity. These results suggest the possibility of translation factors being redox-regulated via Trx in vivo.