• 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.

• Molecules and Cells
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• v.43 no.6
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• pp.551-571
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• 2020

Nuclear receptor-related 1 (Nurr1) protein has been identified as an obligatory transcription factor in midbrain dopaminergic neurogenesis, but the global set of human NURR1 target genes remains unexplored. Here, we identified direct gene targets of NURR1 by analyzing genome-wide differential expression of NURR1 together with NURR1 consensus sites in three human neural stem cell (hNSC) lines. Microarray data were validated by quantitative PCR in hNSCs and mouse embryonic brains and through comparison to published human data, including genome-wide association study hits and the BioGPS gene expression atlas. Our analysis identified ~40 NURR1 direct target genes, many of them involved in essential protein modules such as synapse formation, neuronal cell migration during brain development, and cell cycle progression and DNA replication. Specifically, expression of genes related to synapse formation and neuronal cell migration correlated tightly with NURR1 expression, whereas cell cycle progression correlated negatively with it, precisely recapitulating midbrain dopaminergic development. Overall, this systematic examination of NURR1-controlled regulatory networks provides important insights into this protein's biological functions in dopamine-based neurogenesis.

• Korean Journal of Animal Reproduction
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• v.18 no.4
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• pp.299-307
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• 1995

The goal of cell stem cell technology is to produce a viable and genetically normal animal. To achieve this goal various laboratories have followed 2 different pathways beginning with either the culture of 1) single or pooled ICMs grown with or without a feeder layer or 2) single or pooled 16-20 cell stage embryos grown with a feeder layer. Also, thus far embryonic cell cultures or lines have been established by several methods including loose suspension culture for short-term cultures and more commonly murine or bovine fibroblast feeder layers for long-term culture. Pluripotent lines have been derived from 16-cell through blastocyst inner cell mass stages. The efficiency of establishing cell lines and cell proliferation apper to be affected by the number of cells or embryos starting the line. Most attempts to produce offspring from long term STO cell feeder layer cultured ICM or morulae derived ES cells have resulted in pregnancy failure in the first trimester when ES cells were used in cuclear transfer or have failed to retain ES cells in the progeny produced by chimerization. The exception is 1 chimeric fetus from use of morula ES cells in the chimerization with early embryonic cells. There is much to be learned yet about ES cell culture requirements for maintenance of totipotency. If bovine ES cell lines loose imprinting pattern and totipotency with long-term culture and passage as suggested for mouse ES cells, we may be limited to the use of short-term cultures for multiplication of embryos and efficient production of transgenic animals. No bovine ES cell system has yet met all of the criteria indicated for a totipotent ES cell line.

• 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.

• Korean Journal of Animal Reproduction
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• v.25 no.1
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• pp.71-77
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• 2001

This experiment was designed to improve the production efficiency of germ-line chimeric mice from phospholipase C (PLC)-$\beta$3 or peroxiredoxin (Prx) E -targeted ($\Delta$) ES cells by the investigating the manipulation conditions and characteristics of Jl ES cells. Four targeting clones were isolated to investigate the karyotypical and morphological stability prior to injection. All clones ($\Delta$PLC$\beta$-3 C3, $\Delta$Prx II C3, C10 and I5) showed more than 80% euploidism, however, most of $\Delta$PLC$\beta$-3 C3 clones were extensively differentiated compared to the other clones. Nine of 13 $\Delta$Prx II chimeras appeared to have at least 80% chimerism, whereas $\Delta$PLC$\beta$-3 C3 chimeras had 20% chimerism at most. Therefore, the morphological stability of ES cells under stable euploidism might mainly affect the production rate of high-coat chimeric mice. To increase the collection rate of injectable blastocysts (IBs), 5 to 10 week -aged C57BL/6J female mice were sacrificed at 3.5 days post-coitum. Ten week-aged mice were the most optimal IB donors by showing the highest collection rate (2.94/mouse) of injectable blastocysts without increase of non-injectable embryos (0.29/mouse). Foster mothers might be another factor because ICR x C57BL/6J F1 foster mother showed more increased productivity in litter size (2.8 vs. 5.6) and chimera (0 vs. 35.3%) than those of ICR foster mothers. In conclusion, the efficient production of germ-line chimeras mainly depends on the maintenance of ES cell morphology during targeting procedure, and the establishment of manipulation conditions might be a key point to maximize it.

• Reproductive and Developmental Biology
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• v.32 no.2
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• pp.135-140
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• 2008

Recent studies on nuclear transfer and induced pluripotent stem cells have demonstrated that differentiated somatic cells can be returned to the undifferentiated state by reversing their developmental process. These epigenetically reprogrammed somatic cells may again be differentiated into various cell types, and used for cell replacement therapies through autologous transplantation to treat many degenerative diseases. To date, however, reprogramming of somatic cells into undifferentiated cells has been extremely inefficient. Hence, reliable markers to identify the event of reprogramming would assist effective selection of reprogrammed cells. In this study, a transgene construct encoding enhanced green fluorescent protein (EGFP) under the regulation of human Oct4 promoter was developed as a reporter for the reprogramming of somatic cells. Microinjection of the transgene construct into pronuclei of fertilized mouse eggs resulted in the emission of green fluorescence, suggesting that the undifferentiated cytoplasmic environment provided by fertilized eggs induces the expression of EGFP. Next, the transgene construct was introduced into human embryonic fibroblasts, and the nuclei from these cells were transferred into enucleated porcine oocytes. Along with their in vitro development, nuclear transfer embryos emitted green fluorescence, suggesting the reprogramming of donor nuclei in nuclear transfer embryos. The results of the present study demonstrate that expression of the transgene under the regulation of human Oct4 promoter coincides with epigenetic reprogramming, and may be used as a convenient marker that non-invasively reflects reprogramming of somatic cells.

• Journal of Embryo Transfer
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• v.30 no.4
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• pp.299-303
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• 2015

KO mice provide an excellent tool to determine roles of specific genes in biomedical filed. Traditionally, knockout mice were generated by homologous recombination in embryonic stem cells. Recently, engineered nucleases, such as zinc finger nuclease, transcription activator-like effector nuclease and clustered regularly interspaced short palindromic repeats (CRISPR), were used to produce knockout mice. This new technology is useful because of high efficiency and ability to generate biallelic mutation in founder mice. Until now, most of knockout mice produced using engineered nucleases were C57BL/6 strain. In the present study we used CRISPR-Cas9 system to generate knockout mice in FVB strain. We designed and synthesized single guide RNA (sgRNA) of CRISPR system for targeting gene, Abtb2. Mouse zygote were obtained from superovulated FVB female mice at 8-10 weeks of age. The sgRNA was injected into pronuclear of the mouse zygote with recombinant Cas9 protein. The microinjected zygotes were cultured for an additional day and only cleaved embryos were selected. The selected embryos were surgically transferred to oviduct of surrogate mother and offsprings were obtained. Genomic DNA were isolated from the offsprings and the target sequence was amplified using PCR. In T7E1 assay, 46.7% among the offsprings were founded as mutants. The PCR products were purified and sequences were analyzed. Most of the mutations were founded as deletion of few sequences at the target site, however, not identical among the each offspring. In conclusion, we found that CRISPR system is very efficient to generate knockout mice in FVB strain.

• Reproductive and Developmental Biology
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• v.29 no.1
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• pp.19-24
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• 2005

The low density lipoprotein receptor-related protein 5 (LRP5) highly expressed in many tissues, including hepatocytes and pancreatic beta cells, can bind to apolipoprotein E. To evaluate in vivo roles of LRP5, we generated LRP5-deficient mice. LRP5 genomic DNA was isolated from TT2 embryonic stem (ES) cells. Targeting vector was constructed to disrupt an exon 18 of the mouse LRP5 gene and transfected into ES cells. Three homologous recombinants at LRP5 locus were identified from 178 G418-resistant clones. Chimeric males generated by morula aggregation technique were mated to C57BL/6 female mice. After achieving germ-line transmission, LRP5+/- females were crossed with LRP5+/- males to obtain LRP5-deficient mice. One line of mice lacking LRP5 gene was confirmed by Southern blotting. Such knock-out mice may serve as an effective animal model to study in vivo function of LRP5 gene.

• Asian-Australasian Journal of Animal Sciences
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• v.31 no.3
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• pp.449-456
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• 2018

Objective: Nanog, a homeodomain protein, has been investigated in humans and mice using embryonic stem cells (ESCs). Because of the limited availability of ESCs, few studies have reported the function and role of Nanog in porcine ESCs. Therefore, in this study, we investigated the location of the porcine Nanog chromosome and its basal promoter activity, which might have potential applications in development of ESCs specific marker as well as understanding its operating systems in the porcine. Methods: To characterize the porcine Nanog promoter, the 5'-flanking region of Nanog was isolated from cells of mini-pig ears. BLAST database search showed that there are two porcine Nanog genomic loci, chromosome 1 and 5, both of which contain an exon with a start codon. Deletion mutants from the 5'-flanking region of both loci were measured using the Dual-Luciferase Reporter Assay System, and a fluorescence marker, green fluorescence protein. Results: Promoter activity was detected in the sequences of chromosome 5, but not in those of chromosome 1. We identified the sequences from -99 to +194 that possessed promoter activity and contained transcription factor binding sites from deletion fragment analysis. Among the transcription factor binding sites, a Sp1 was found to play a crucial role in basal promoter activity, and point mutation of this site abolished its activity, confirming its role in promoter activity. Furthermore, gel shift analysis and chromatin immunoprecipitation analysis confirmed that Sp1 transcription factor binds to the Sp1 binding site in the porcine Nanog promoter. Taken together, these results show that Sp1 transcription factor is an essential element for porcine Nanog basal activity the same as in human and mouse. Conclusion: We showed that the porcine Nanog gene is located on porcine chromosome 5 and its basal transcriptional activity is controlled by Sp1 transcription factor.

• Development and Reproduction
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• v.12 no.1
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• pp.1-8
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• 2008

Specific protocols to increase the differentiation of neuronal cells from embryonic stem (ES) cells have been well established, such as retinoic acid induction and lineage selection of neuronal cells. For the neuropathological studies, ES-derived neurons (ES neurons) must show normal physiological characteristics related to cell death and survival and should be maintained in vitro for a sufficient time to show insults-specific cell death without spontaneous death. When mouse ES cells were plated onto astrocytes monolayer after retinoic acid induction, most ES cells differentiated into neuronal cells, which were confirmed by the presence of specific neuronal markers, and the cultures were viable for at least four weeks. When these cultures were examined for vulnerability to glutamate excitotoxicity, ES neurons were vulnerable to excitotoxic insults mediated by agonist-specific receptors. The vulnerability to excitotoxic death increased with developmental age of ES neurons in vitro. Specific receptors for Neurotrophin and GDNF family ligands were present in ES neurons. GDNF and NT-3 could modulate the survival and excitotoxic vulnerability of ES neurons. The vulnerability and resistance to toxic insults, which are essential requirements of model culture systems for neuropathological studies, make ES neurons to a useful model culture system. Especially ES cell are highly amenable to genetic modification unlikely to primary neuronal cells, which will give us a chance to answer more complicated neurophysiological questions. Recently there was an outstanding attempt to explore the cellular toxicity using human ES cells (Schrattenholz & Klemm, 2007) and it suggested that ES cells could be a new model system for neurophysiological studies soon and go further a large-scale screening system for pharmacological compounds in the future.