• Asian-Australasian Journal of Animal Sciences
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• v.21 no.5
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• pp.745-753
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• 2008

Gene-manipulated mice were discovered for the first time about a quarter century ago. Since then, numerous sophisticated technologies have been developed and applied to answer key questions about the fundamental roles of the genes of interest. Functional genomics can be characterized into gain-of-function and loss-of-function, which are called transgenic and knock-out studies, respectively. To make transgenic mice, the most widely used technique is the microinjection of transgene-containing vectors into the embryonic pronucleus. However, there are critical drawbacks: namely position effects, integration of unknown copies of a foreign gene, and instability of the foreign DNA within the host genome. To overcome these problems, the ROSA26 locus was used for the knock-in site of a transgene. Usage of this locus is discussed for the gain of function study as well as for several brilliant approaches such as conditional/inducible transgenic system, reproducible/inducible knockdown system, specific cell ablation by Cre-mediated expression of DTA, Cre-ERTM mice as a useful tool for temporal gene regulation, MORE mice as a germ line delete and site specific recombinase system. Techniques to make null mutant mice include complicated steps: vector design and construction, colony selection of embryonic stem (ES) cells, production of chimera mice, confirmation of germ line transmission, and so forth. It is tedious and labor intensive work and difficult to approach. Thus, it is not readily accessible by most researchers. In order to overcome such limitations, technical breakthroughs such as reporter knock-in and gene knock-out system, production of homozygous mutant ES cells from a single targeting vector, and production of mutant mice from tetraploid embryos are developed. With these upcoming progresses, it is important to consider how we could develop these systems further and expand to other animal models such as pigs and monkeys that have more physiological similarities to humans.

• Journal of Embryo Transfer
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• v.18 no.3
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• pp.243-248
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• 2003

Sperrnatogenesis, the process by which the male germ-line stem cells(GSCs; type A spermatogonia) divide and differentiate to produce the mature spermatozoa, occurs in the seminiferous tubules of the testis. The GSCs proliferate actively to produce two types of cells: other GSCs and differentiating spermatogonia. GSCs have unipotentcy, devoted solely to the generation of sperm. The function of GSCs has broad implications for development, disease, and evolution. Spermatogenesis is fundamental for propagation of species and the defects of this system can result in infertility or disease. The ability to identify, isolate, culture, and alter GSCs will allow powerful new approaches in animal transgenesis and human gene therapy relating to infertility. Until recently, research on stem cells in the testis has been limited because of technical difficulties in isolating and identifying these cell populations. Here, we were trying to find out optimal conditions for in vitro culture of GSCs for identifying and isolating GSCs. We collected mouse GSCs from 3-days old mouse by two-step enzyme digestion method. GSCs were plated and grown on mouse embryonic fibroblasts in Dulbecco's modified Eagle's medium (DMEM) containing 15％ fatal bovine serum, 10 mM 2-mercaptoethanol, 1％ non-essential amino acids, 1 ng/$m\ell$ bFGF, 10 $\mu$M forskolin, 1500 U/$m\ell$ human recombinant leukemia inhibitory factor (LIF). Over a period 3∼5 days, GSCs gave rise to large multicellular colonies resembling those of mouse pluripotent stem cells. After 5th passages, cells within the colonies continued to be alkaline phosphatase and Oct-4 positive and tested positive against a panel of two immunological markers(Integrin $\alpha$ 6 and Integrin $\beta$ 1) that have been recognized generally to characterize GSCs. SSEA-1, SSEA-3, and SSEA-4 also showed positive signals. Based on our data, these GSCs-derived cultures meet the criteria for GSCs itself and even other pluripotent stem cells. We reported here the establishment of in vitro cultures from mouse male GSCs.

• Journal of Microbiology and Biotechnology
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• v.22 no.12
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• pp.1790-1794
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• 2012

Recently, it has been suggested that $p27^{Kip1}$, the cell cycle regulatory protein, plays a pivotal role in the progression of normal differentiation in murine embryonic stem (mES) cells. In the current study, we investigated the role of $p27^{Kip1}$ in the regulation of differentiation and apoptotic induction using Western blotting, quantitative real-time RT-PCR, and small interfering RNA (siRNA) assays and confocal laser scanning microscopic analysis of H9 human ES (hES) cells and H9-derived embryoid bodies (EBs) grown for 10 ($EB_{10}$) and 20 days ($EB_{20}$). Our results demonstrate that the proteins $p27^{Kip1}$ and cyclin D3 are strongly associated with cellular differentiation, and, for the first time, show that up-regulation of $p27^{Kip1}$ protects hES cells from inducing differentiation-associated and caspase 3-dependent apoptosis.

• International Journal of Oral Biology
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• v.35 no.4
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• pp.209-214
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• 2010

Cells that have endogenous multipotent properties can be used as a starting source for the generation of induced pluripotent cells (iPSC). In addition, small molecules associated with epigenetic reprogramming are also widely used to enhance the multi- or pluripotency of such cells. Skinderived precursor cells (SKPs) are multipotent, sphereforming and embryonic neural crest-related precursor cells. These cells can be isolated from a juvenile or adult mammalian dermis. SKPs are also an efficient starting cell source for reprogramming and the generation of iPSCs because of the high expression levels of Sox2 and Klf4 in these cells as well as their endogenous multipotency. In this study, valproic acid (VPA), a histone deacetylase (HDAC) inhibitor, was tested in the generation of iPSCs as a potential enhancer of the reprogramming potential of SKPs. SKPs were isolated from the back skins of 5-6 week old C57BL/6 X DBA/2 F1 mice. After passage 3, the SKPs was treated with 2 mM of VPA and the quantitative real time RT-PCR was performed to quantify the expression of Oct4 and Klf4 (pluripotency specific genes), and Snai2 and Ngfr (neural crest specific genes). The results show that Oct4 and Klf4 expression was decreased by VPA treatment. However, there were no significant changes in neural crest specific gene expression following VPA treatment. Hence, although VPA is one of the most potent of the HDAC inhibitors, it does not enhance the reprogramming of multipotent skin precursor cells in mice.

• Journal of Life Science
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• v.19 no.4
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• pp.549-552
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• 2009

Genetic mutations by gene fusion result from chromosomal rearrangement, trans-splicing, and intergenic splicing. Trans-splicing is a phenomenon in which two pre-mRNAs grow together into one. We analyzed the trans-splicing products in embryonic stem cells. By using bioinformatic tools, 70 trans-splicing transcripts were identified. They are classified into 6 types according to fusion pattern: 5'UTR-5'UTR, 5'UTR-3'UTR, 3'UTR-3'UTR, 5'UTR-CDS, 3'UTR-CDS, CDS-CDS. The fusion products are more abundant in CDS regions than in UTR regions, which contain multiple intron numbers. Chromosome analysis showing gene fusion via trans-splicing indicated that chromosomes 17 and 19 were activated. These data are of great use for further studies in relation to fusion genes and human diseases.

• The Korean Society of Law and Medicine
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• v.12 no.1
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• pp.227-255
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• 2011

Recently, many nations said "yes" to human embryonic stem cell research, signing an executive order to permit funding for the research in the mame of achieving health and life of humankind. Human Embryo Research is permitted by our Bioethics & Biosafety Act. But, illegal research cannot be divorced from civil liability since it requires the destruction of eggs of fertilized eggs and personal rights of embryo-creator. After all, though we allow to do research embryo, we should control the capacity of abuse of embryo research for embryo-creator. If research violate the law(Bioethics & Biosafety Act or Civil Law, etc), it comes to a delict by pecuniary loss and non-pecuniary loss. When it comes to pecuniary loss, Human Embryo is not body but special property. Supreme Court maintained a stance that mental suffering is generally deemed as compensable for damages for the loss of property where a person's property right is invaded by a tort or non-performance of obligation. Thus, where mental suffering occurs, which cannot be compensated by recovery of property losses, the situation must be a special circumstance and the injured could claim consolation money for such losses only if the offender knew or would have known of such special circumstances(Supreme Court Decision 96Da31574 delivered on Nov, 26, 1996, etc.). That is to say, Supreme Court regards mental suffering through person's property right invaded by a tort as damages that have arisen through special circumstances. According to Civil law article 393 (2), the injured could claim consolation money for such losses only if only if the offender had foreseen or could have foreseen such circumstances. Also our court will solve through damages for non-pecuniary loss by complementary function of consolation money in that pecuniary loss could be difficult to valuate.

• Laboraroty Animal Research
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• v.34 no.4
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• pp.279-287
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• 2018

Placenta specific 8 (PLAC8, also known as ONZIN) is a multi-functional protein that is highly expressed in the intestine, lung, spleen, and innate immune cells, and is involved in various diseases, including cancers, obesity, and innate immune deficiency. Here, we generated a Plac8 knockout mouse using the CRISPR/Cas9 system. The Cas9 mRNA and two single guide RNAs targeting a region near the translation start codon at Plac8 exon 2 were microinjected into mouse zygotes. This successfully eliminated the conventional translation start site, as confirmed by Sanger sequencing and PCR genotyping analysis. Unlike the previous Plac8 deficient models displaying increased adipose tissue and body weights, our male Plac8 knockout mice showed rather lower body weight than sex-matched littermate controls, though the only difference between these two mouse models is genetic context. Differently from the previously constructed embryonic stem cell-derived Plac8 knockout mouse that contains a neomycin resistance cassette, this knockout mouse model is free from a negative selection marker or other external insertions, which will be useful in future studies aimed at elucidating the multi-functional and physiological roles of PLAC8 in various diseases, without interference from exogenous foreign DNA.

• Laboraroty Animal Research
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• v.34 no.4
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• pp.257-263
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• 2018

Trefoil factor 1 (TFF1, also known as pS2) is strongly expressed in the gastrointestinal mucosa and plays a critical role in the differentiation of gastric glands. Since approximately 50% of all human gastric cancers are associated with decreased TFF1 expression, it is considered a tumor suppressor gene. Tff1 deficiency in mice results in histological changes in the antral and pyloric gastric mucosa, with severe hyperplasia and dysplasia of epithelial cells, resulting in the development of antropyloric adenoma. Here, we generated Tff1-knockout (KO) mice, without a neomycin resistant ($Neo^R$) cassette, using the clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease 9 (CRSIPR/Cas9) system. Though our Tff1-KO mice showed phenotypes very similar to the previous embryonic stem (ES)-cell-based KO mice, they differed from the previous reports in that a reduction in body weight was observed in males. These results demonstrate that these newly established Tff1-KO mice are useful tools for investigating genetic and environmental factors influencing gastric cancer, without the effects of artificial gene insertion. Furthermore, these findings suggest a novel hypothesis that Tff1 expression influences gender differences.

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2003.10a
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• pp.60-60
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• 2003

Embryonic stem (ES) cells have property of self-renewal and can differentiate into the cells of all three primary germ layers. Recently, many growth factors, alteration of culture condition and gene modifications have been used to differentiate mouse and human ES cells into specific cell types. This study was performed to evaluate the differentiation protocol for human ES cells to the endodermal lineage cells. Human ES cells (Miz-hESl ) were cultured on STO feeder layer mitotically inactivated with mitemycin C, and embryoid bodies (EBs) were formed by suspension culture. Differentiation protocol of EBs consisted of three steps: stage I, culture of EBs for 6 days with ITSFn medium; stage II, culture of stage I cells for 8 days with N2 medium ; stage III, culture of stage II cells for 22 days with N2 medium. mRNA levels of the endodermal lineage differentiation genes were analyzed by semi- quantitative RT-PCR. The Oct-4 expression, a marker of the pluripotent state, was detected in undifferentiated human ES cells but progressively decreased after EBs formation. Differentiating human ES cells expressed marker genes of endodermal differentiation and pancreatic islet cells. GATA4, a-fetoprotein, Glut-2, and Ngn3 were expressed in all stages. However, albumin and insulin were expressed in only stage III cells. The human ES cells can be differentiated into endodermal lineage cells by multiple step culture system using various supplements. We are developing the more effective protocols for guided differentiation of human ES cells.

• Asian Pacific Journal of Cancer Prevention
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• v.13 no.11
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• pp.5469-5475
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• 2012

Background and Objectives: Acute lymphoblastic leukemia (ALL) is a complex genetic disease involving many fusion oncogenes (FO) having prognostic significance. The frequency of various FO can vary in different ethnic groups, with important implications for prognosis, drug selection and treatment outcome. Method: We studied fusion oncogenes in 101 pediatric ALL patients using interphase FISH and RT-PCR, and their associations with clinical features and treatment outcome. Results: Five most common fusion genes i.e. BCR-ABL t (22; 9), TCF3-PBX1 (t 1; 19), ETV6-RUNX1 (t 12; 21), MLL-AF4 (t 4; 11) and SIL-TAL1 (del 1p32) were found in 89/101 (88.1%) patients. Frequency of BCR-ABL was 44.5% (45/101). BCR-ABL positive patients had a significantly lower survival ($43.7{\pm}4.24$ weeks) and higher white cell count as compared to others, except patients with MLL-AF4. The highest relapse-free survival was documented with ETV6-RUNX1 (14.2 months) followed closely by those cases in which no gene was detected (13.100). RFS with BCR-ABL, MLL-AF4, TCF3-PBX1 and SIL-TAL1 was less than 10 months (8.0, 3.6, 5.5 and 8.1 months, respectively). Conclusions: This is the first study from Pakistan correlating molecular markers with disease biology and treatment outcome in pediatric ALL. It revealed the highest reported frequency of BCR-ABL FO in pediatric ALL, associated with poor overall survival. Our data indicate an immediate need for incorporation of tyrosine kinase inhibitors in the treatment of BCR-ABL+ pediatric ALL in this population and the development of facilities for stem cell transplantation.