• Development and Reproduction
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• v.14 no.4
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• pp.215-223
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• 2010

The construction of transgenic mouse using embryonic stem (ES) cells has been crucial in the functional studies of gene on mouse genome. Gene knockout mice have been powerful for elucidating the function of genes as well as a research model for human diseases. Gene targeting and gene trapping mathods have been the representative technologies for making the knockout mice by using ES cells. Since the gene targeting and the gene trapping methods were independently developed about 20 years ago, it's efficiency and productivity has been improved with a advance of molecular biology. Conventional gene targeting method has been changes to high throughput conditional gene targeting. The combination of the advantage of gene targeting and gene tapping elements allows to extend a spectrum of gene trapping and to improve the efficiency of gene targeting. These advance should be able to produce the mutant with various phenotype to target a certain gene, and in postgenome era they have served as crucial research tools in understanding the functional study of whole genome in mouse.

• Proceedings of the KSAR Conference
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• 2004.06a
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• pp.193-193
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• 2004

Gene targeting is an in situ manipulation of endogenous gene with precise manner by the introduction of exogenous DNA. The process of gene targeting involves a homologous recombination reaction between the targeted genomic sequence and an exogenous targeting vector. In elucidating the function of many genes, gene targeting has become the most important method of choice. (omitted)

• KSBB Journal
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• v.25 no.3
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• pp.215-222
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• 2010

Monogenic diseases are resulted from modifications in a single gene of human cells. Because their treatment with pharmacological medicine have a temporary effect, continuous nursing care and retreatment are required. Gene therapy, gene targeting and induced pluripotent stem cell (iPSC) are considered permanent treatment methods of them. In gene therapy, however, retroviral vectors that have potential toxicity caused by random insertion of harmful virus are used as vehicles for transferring genetic materials. On the other hand, gene targeting could replace and remove the modified gene though homologous recombination (HR) induced by site-specific endonucleases. This short review provides a brief overview on the recently tailored endonucleses with high selectivity for HR.

• Reproductive and Developmental Biology
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• v.38 no.2
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• pp.71-77
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• 2014

The specific genetic modification in porcine somatic cells by gene targeting has been very difficult because of low efficiency of homologous recombination. To improve gene targeting, we designed three kinds of knock-out vectors with ${\alpha}1,3$-galactosyltransferase gene (${\alpha}1,3$-GT gene), DT-A/pGT5'/neo/pGT3', DT-A/NLS/pGT5'/neo/pGT3' and pGT5'/neo/ pGT3'/NLS. The knock-out vectors consisted of a 4.8-kb fragment as the 5' recombination arm (pGT5') and a 1.9-kb fragment as the 3' recombination arm (pGT3'). We used the neomycin resistance gene (neo) as a positive selectable marker and the diphtheria toxin A (DT-A) gene as a negative selectable marker. These vectors have a neo gene insertion in exon 9 for inactivation of ${\alpha}1,3$-GT locus. DT-A/pGT5'/neo/pGT3' vector contain only positive-negative selection marker with conventional targeting vector. DT-A/NLS/pGT5'/neo/pGT3' vector contain positive-negative selection marker and NLS sequences in upstream of 5' recombination arm which enhances nuclear transport of foreign DNA into bovine somatic cells. pGT5'/neo/pGT3'/NLS vector contain only positive selection marker and NLS sequence in downstream of 3' recombination arm, not contain negative selectable marker. For transfection, linearzed vectors were introduced into porcine ear fibroblasts by electroporation. After 48 hours, the transfected cells were selected with $300{\mu}g/ml$ G418 during 12 day. The G418-resistant colonies were picked, of which 5 colonies were positive for ${\alpha}1,3$-GT gene disruption in 3' PCR and southern blot screening. Three knock-out somatic cells were obtained from DT-A/NLS/ pGT5'/neo/pGT3' knock-out vector. Thus, these data indicate that gene targeting vector using nuclear localization signal and negative selection marker improve targeting efficiency in porcine somatic cells.

• BMB Reports
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• v.37 no.1
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• pp.122-132
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• 2004

The rapid development and characterization of the mouse genome sequence, coupled with comparative sequence analysis of human, has been paralleled by a reinforced enthusiasm for mouse functional genomics. The way to uncover the in vivo function of genes is to analyze the phenotypes of the mutant animals. From this standpoint, the mouse is a suitable and valuable model organism in the studies of functional genomics. Therefore, there have been enormous efforts to enrich the list of the mutant mice. Such a trend emphasizes the random mutagenesis, including ENU mutagenesis and gene-trap mutagenesis, to obtain a large stock of mutant mice. However, since various mutant alleles are needed to precisely characterize the role of a gene in vivo, mutations should be designed. The simplicity and utility of transgenic technology can satisfy this demand. The combination of RNA interference with transgenic technology will provide more opportunities for researchers. Nevertheless, gene targeting can solely define the in vivo function of a gene without a doubt. Thus, transgenesis and gene targeting will be the major strategies in the field of functional genomics.

• Journal of Plant Biotechnology
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• v.42 no.3
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• pp.154-160
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• 2015

The plant breeding technology was developed with genetic engineering. Many researchers and breeders have turned from traditional breeding to molecular breeding. Genetically modified organisms (GMO) were developed via molecular breeding technology. Currently, molecular breeding technologies facilitate efficient plant breeding without introducing foreign genes, in virtue by of gene editing technology. Gene targeting (GT) via homologous recombination (HR) is one of the best gene editing methods available to modify specific DNA sequences in genomes. GT utilizes DNA repair pathways. Thus, DNA repair systems are controlled to enhance HR processing. Engineered sequence specific endonucleases were applied to improve GT efficiency. Engineered sequence specific endonucleases like the zinc finger nuclease (ZFN), TAL effector nuclease (TALEN), and CRISPR-Cas9 create DNA double-strand breaks (DSB) that can stimulate HR at a target site. RecQl4, Exo1 and Rad51 are effectors that enhance DSB repair via the HR pathway. This review focuses on recent developments in engineered sequence specific endonucleases and ways to improve the efficiency of GT via HR effectors in plants.

• BMB Reports
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• v.45 no.12
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• pp.686-692
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• 2012

Phenotypic analysis of gene-specific knockout (KO) mice has revolutionized our understanding of in vivo gene functions. As the use of mouse embryonic stem (ES) cells is inevitable for conventional gene targeting, the generation of knockout mice remains a very time-consuming and expensive process. To accelerate the large-scale production and phenotype analyses of KO mice, international efforts have organized global consortia such as the International Knockout Mouse Consortium (IKMC) and International Mouse Phenotype Consortium (IMPC), and they are persistently expanding the KO mouse catalogue that is publicly available for the researches studying specific genes of interests in vivo. However, new technologies, adopting zinc-finger nucleases (ZFNs) or Transcription Activator-Like Effector (TALE) Nucleases (TALENs) to edit the mouse genome, are now emerging as valuable and effective shortcuts alternative for the conventional gene targeting using ES cells. Here, we introduce the recent achievement of IKMC, and evaluate the significance of ZFN/TALEN technology in mouse genetics.

• Korean Journal of Cleft Lip And Palate
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• v.11 no.2
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• pp.65-70
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• 2008

Cleft lip and/or palate are common birth defects in humans and the causes including multiple genetic and environmental factors are complex. Combinations of genetic, biochemical, and embryological approaches in the laboratory mice are used to investigate the molecular mechanisms underlying normal craniofacial development and the congenital craniofacial malformations including cleft lip and/or palate. Both forward and reverse genetic approaches are used. The forward genetic approach involves identification of causative genes and molecular pathways disrupted by uncharacterized mutations that cause craniofacial malformations including cleft lip and/or cleft palate. The reverse genetic approach involves generation and analyses of mice carrying null or conditional mutations using the Cre-loxP mediated gene targeting techniques.

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2003.10a
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• pp.86-86
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• 2003

Research has been in progress for more than a decade to production of useful proteins by genetic modification in cattle. However, the levels of protein production in transgenic cattle have been reported very low. To enhance protein production in transgenic animal, we tried homologous recombination to donor cells for production of transgenic clone cattle through nuclear transfer procedure. Thus, we constructed the two targeting vectors of human thrombopoietin (TPO) at bovine $\beta$-casein locus using homologous recombination with 13.6 kb and 9.6 kb homology. In two targeting vectors, positive selection was through the neomycin resistance gene and negative selection was by the diphtheria toxin (DT). Gene targeting was attempted in bovine embryonic fibroblasts (bEF) and bovine ear skin fibroblasts (bESF). To determine the most appropriate concentration of neomycin for bEF and bESF, G4l8 resistance was confirmed by culturing the cells in various concentrations of the drug and both of the cells were optimally selected at $900 \mu g/ml$ of neomycin. The transfected bEF and bESF by the targeting vectors were colonized efficiently at the ratio of DNA to transfection reagent such as $4 \mu g$:2 ${mu}ell$ and $1 \mu g$:$2 \mu l$. Comparing number of healthy clones from passage 4 to passage 8, bESF (17%) persist in culture for much longer than bEF (6%). The two gene-targeted bESF clones of 30 random-integrated clones with 9.6 kb homology length were confirmed, however, nothing was out of 72 random integration clones with 13.6 kb homology length, The DT also worked more efficiently in clones transfected with the vector of 9.6 kb homology length. Our data suggests that the choice of donor cell for long culture period should be considered to obtain targeted cell clone, and the gene-targeting frequency and the DT working efficiency are dependent on the length of target homology.

• Toxicological Research
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• v.17
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• pp.289-292
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• 2001

Gene targeting allows precise, predetermined changes to be made in a chosen gene in the mouse genome. To date, targeting has been used most often for generation of animals completely lacking the product of a gene of interest. Models of essential hypertension have been produced by mutated genes relating renin angiotensin system. The most significant contribution to understanding the genetic etiology of essential hypertension is probably the demonstration that discrete alterations in the expression of a variety of different genes can individually cause changes in the blood pressures of mice, even when the mice have all their compensatory mechanisms intact. These effects are readily detected in animals having moderate decreases in gene function due to heterozygosity for gene disruptions or modest increases due to gene duplication. As a species the mouse is highly resistant to atherosclerosis. However. through induced mutations it has been possible to develop lines oj mice that are deficient in apolipoprotein E, a ligand important in lipoprotein clearance, develop atherosclerotic lesions resembling those observed in humans. The atherosclerotic lesions in apoE-deficient mice have been well characterized, and they resemble human lesions in their sites of predilection and progression to the fibroproliferative stage. Other promising models are mice that are deficient in the low-density lipoprotein receptor. Considerable work still remains to be done in dissecting out in a rigorous manner the effects of alterations in single genes on the induction or progression of atherosclerosis and on the control of blood pressures. Perhaps even more exciting is the opportunity now becoming available to breed animals in which the effects oj precise differences in more than one gene can be studied in combination.