• Asian-Australasian Journal of Animal Sciences
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• v.30 no.5
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• pp.743-748
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• 2017

Objective: Based on rapid advancement of genetic modification techniques, genomic editing is expected to become the most efficient tool for improvement of economic traits in livestock as well as poultry. In this study, we examined and verified the nickase of mutated CRISPR-associated protein 9 (Cas9) to modulate the specific target gene in chicken DF1 cells. Methods: Chicken myostatin which inhibits muscle cell growth and differentiation during myogenesis was targeted to be deleted and mutated by the Cas9-D10A nickase. After co-transfection of the nickase expression vector with green fluorescent gene (GFP) gene and targeted multiplex guide RNAs (gRNAs), the GFP-positive cells were sorted out by fluorescence-activated cell sorting procedure. Results: Through the genotyping analysis of the knockout cells, the mutant induction efficiency was 100% in the targeted site. Number of the deleted nucleotides ranged from 2 to 39 nucleotide deletion. There was no phenotypic difference between regular cells and knockout cells. However, myostatin protein was not apparently detected in the knockout cells by Western blotting. Additionally, six off-target sites were predicted and analyzed but any non-specific mutation in the off-target sites was not observed. Conclusion: The knockout technical platform with the nickase and multiplex gRNAs can be efficiently and stablely applied to functional genomics study in poultry and finally adapted to generate the knockout poultry for agribio industry.

• The Korean Journal of Physiology and Pharmacology
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• v.24 no.3
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• pp.233-240
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• 2020

Autophagy regulators are often effective as potential cancer therapeutic agents. Here, we investigated paclitaxel sensitivity in cells with knockout (KO) of ATG5 gene. The ATG5 KO in multidrug resistant v-Ha-ras-transformed NIH 3T3 cells (Ras-NIH 3T3/Mdr) was generated using the CRISPR/Cas9 technology. The qPCR and LC3 immunoblot confirmed knockout of the gene and protein of ATG5, respectively. The ATG5 KO restored the sensitivity of Ras-NIH 3T3/Mdr cells to paclitaxel. Interestingly, ATG5 overexpression restored autophagy function in ATG5 KO cells, but failed to rescue paclitaxel resistance. These results raise the possibility that low level of resistance to paclitaxel in ATG5 KO cells may be related to other roles of ATG5 independent of its function in autophagy. The ATG5 KO significantly induced a G₂/M arrest in cell cycle progression. Additionally, ATG5 KO caused necrosis of a high proportion of cells after paclitaxel treatment. These data suggest that the difference in sensitivity to paclitaxel between ATG5 KO and their parental MDR cells may result from the disparity in the proportions of necrotic cells in both populations. Thus, our results demonstrate that the ATG5 KO in paclitaxel resistant cells leads to a marked G₂/M arrest and sensitizes cells to paclitaxel-induced necrosis.

• Journal of Genetic Medicine
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• v.20 no.2
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• pp.39-45
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• 2023

As advanced sequencing technologies continue to uncover an increasing number of variants in genes associated with human genetic diseases, there is a growing demand for systematic approaches to assess the impact of these variants on human development, health, and disease. While in silico analyses have provided valuable insights, it is essential to complement these findings with model organism studies to determine the functional consequences of genetic variants in vivo. Drosophila melanogaster is an excellent genetic model for such functional studies due to its efficient genetic technologies, high gene conservation with humans, accessibility to mutant fly resources, short life cycles, and cost-effectiveness. The traditional GAL4-UAS system, allowing precise control of gene expression through binary regulation, is frequently employed to assess the effects of monoallelic variants. Recombinase medicated cassette exchange or CRISPR-Cas9-mediated GAL4 insertion within coding introns or substitution of gene body with Kozak-Gal4 result in the loss-of-function of the target gene. This GAL4 insertion strategy also enables the expression of reference complementary DNA (cDNA) or cDNA carrying genetic variants under the control of endogenous regulatory cis elements. Furthermore, the CRISPR-Cas9-directed tissue-specific knockout and cDNA rescue system provides the flexibility to investigate candidate variants in a tissue-specific and/or developmental-timing dependent manner. In this review, we will delve into the diverse genetic techniques available in Drosophila and their applications in diagnosing and studying numerous undiagnosed diseases over the past decade.

• Animal Bioscience
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• v.36 no.2_spc
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• pp.333-338
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• 2023

Genetic modification enables modification of target genes or genome structure in livestock and experimental animals. These technologies have not only advanced bioscience but also improved agricultural productivity. To introduce a foreign transgene, the piggyBac transposon element/transposase system could be used for production of transgenic animals and specific target protein-expressing animal cells. In addition, the clustered regularly interspaced short palindromic repeat-CRISPR associated protein 9 (CRISPR-Cas9) system have been utilized to generate chickens with knockout of G0/G1 switch gene 2 (G0S2) and myostatin, which are related to lipid deposition and muscle growth, respectively. These experimental chickens could be the invaluable genetic resources to investigate the regulatory pathways and mechanisms of improvement of economic traits such as fat quantity and growth. The gene-edited animals could also be applicable to the livestock industry.

• Horticultural Science & Technology
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• v.30 no.6
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• pp.734-742
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• 2012

To compare RNA interference-mediated gene silencing technique and T-DNA integration for gene function analysis in Chinese cabbage, BrSAMS-knockout (KO) line and BrSAMS-knockdown (KD) line were used. The KO line had lost the function of a Brassica rapa S-adenosylmethionine synthetase (BrSAMS) gene by T-DNA insertion and the KD line had shown down-regulated BrSAMS genes' expression by dsRNA cleavage. From microarray results of the KO and KD lines, genes linked to SAMS such as sterol, sucrose, homogalacturonan biosynthesis and glutaredoxin-related protein, serine/threonine protein kinase, and gibberellin-responsive protein showed distinct differences in their expression levels. Even though one BrSAMS gene in the KO line was broken by T-DNA insertion, gene expression pattern of that line did not show remarkable differences compared to wild type control. However, the KD line obtained by RNAi technique showed prominent difference in its gene expression. Besides, change of polyamine and ethylene synthesis genes directly associated with BrSAMS was displayed much more in the KD line. In the microarray analysis of the KO line, BrSAMS function could not be clearly defined because of BrSAMS redundancy due to the genome triplication events in Brassicaceae. In conclusion, we supposed that gene knock-down method by RNAi silencing is more effective than knock-out method by T-DNA insertion for gene function analysis of polyploidy crops such as Chinese cabbage.

• Animal Bioscience
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• v.35 no.6
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• pp.791-803
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• 2022

Genome/gene-editing (GE) techniques, characterized by a low technological barrier, high efficiency, and broad application among organisms, are now being employed not only in medical science but also in agriculture/veterinary science. Different engineered CRISPR/Cas9s have been identified to expand the application of this technology. In pig production, GE is a precise new breeding technology (NBT), and promising outcomes in improving economic traits, such as growth, lean or healthy meat production, animal welfare, and disease resistance, have already been documented and reviewed. These promising achievements in porcine gene editing, including the Myostatin gene knockout (KO) in indigenous breeds to improve lean meat production, the uncoupling protein 1 (UCP1) gene knock-in to enhance piglet thermogenesis and survival under cold stress, the generation of GGTA1 and CMP-N-glycolylneuraminic acid hydroxylase (CMAH) gene double KO (dKO) pigs to produce healthy red meat, and the KO or deletion of exon 7 of the CD163 gene to confer resistance to porcine reproductive and respiratory syndrome virus infection, are described in the present article. Other related approaches for such purposes are also discussed. The current trend of global regulations or legislation for GE organisms is that they are exempted from classification as genetically modified organisms (GMOs) if no exogenes are integrated into the genome, according to product-based and not process-based methods. Moreover, an updated case study in the EU showed that current GMO legislation is not fit for purpose in term of NBTs, which contribute to the objectives of the EU's Green Deal and biodiversity strategies and even meet the United Nations' sustainable development goals for a more resilient and sustainable agri-food system. The GE pigs generated via NBT will be exempted from classification as GMOs, and their global valorization and commercialization can be foreseen.

• Proceedings of the Korean Nutrition Society Conference
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• 2002.05a
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• pp.29-41
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• 2002

While the sequencing of several genomes was underway, several advanced techniques in genetics, molecular biology and protein chemistry emerged. Within the nutritional sciences, while the focus on nutrition education, epidemiology and public health aspects remains essential; it is crucial to incorporate the new advances in gene and protein discovery in nutritional studies. Nutrition is a discipline that has always integrated social, biochemical and physiological sciences from the studies at the molecule level to studies at the population level. For this reason, nutritionists are in a prime position to readily incorporate the current genomics approaches in nutrition research, All the available analytical techniques can and should be used in modern nutritional sciences. These include genetics, genomics, proteomics and metabolomics which also require integration and use of bioinformatics and computational methods for data analysis and management. These applications will be briefly reviewed with a primary focus on what the genomics and genetics approaches offer to nutritionists. We will use one of our research focus areas to illustrate uses of some of these applications in obesity-hypertension research. Our central hypothesis is that adipose tissue is an endocrine organ that plays a major role in obesity and related hypertension. We are primarily studying the renin angiotensin system (RAS). We provide evidence from our own studies and others for the paracrine as well as endocrine role of adipocyte-derived angiotensin II in adipocyte gene expression, adiposity and blood pressure regulation. Both cell culture studies as well as knockout and transgenic mice models are used to test our hypothesis. Genomics and proteomics technologies are currently developed to complement our physiological and molecular studies on the RAS and for a fine analysis of this system and its function in health and disease.

• Proceedings of the Korean Nutrition Society Conference
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• 2002.06a
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• pp.598-603
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• 2002

While the sequencing of several genomes was underway, several advanced techniques in genetics, molecular biology and protein chemistry emerged. Within the notritional sciences, while the focus on nutrition education, epidemiology and public health aspects remains essential; it is crucial to incorporate the new advances in gene and protein discovery in nutritional studies. Nutrition is a discipline that has always integrated social, biochemical and physiological sciences from the studies at the molecule level to studies at the population level. for this reason, nutritionists are in a prime position to readily incorporate the current genomics approaches in nutrition research. All the available analytical techniques can and should be used in modem nutritional sciences. These include genetics, genomics, proteomics and metabolomics which also require integration and use of bioinformatics and computational methods for data analysis and management. These applications will be briefly reviewed with a primary focus on what the genomics and genetics approaches offer to nutritionists. We will use one of our research focus areas to illustrate uses of some of these applications in obesity-hypertension research. Our central hypothesis is that adipose tissue is an endocrine organ that plays a major role in obesity and related hypertension. We are primarily studying the renin angiotensin system (RAS). We provide evidence from our own studies and others for the paracrine as well as endocrine role of adipocyte-derived angiotensin II in adipocyte gene expression, adiposity and blood pressure regulation. Both cell culture studies as well as knockout and transgenic mice models are used to test our hypothesis. Genomics and proteomics technologies are currently developed to complement our physiological and molecular studies on the RAS and for a fine analysis of this system and its function in health and disease.