• Animal Bioscience
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• 제35권1호
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• pp.126-137
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• 2022

Objective: Efficient gene editing technology is critical for successful knock-in in domestic animals. RAD51 recombinase (RAD51) gene plays an important role in strand invasion during homologous recombination (HR) in mammals, and is regulated by checkpoint kinase 1 (CHK1) and CHK2 genes, which are upstream elements of RAD51 recombinase (RAD51). In addition, mismatch repair (MMR) system is inextricably linked to HR-related pathways and regulates HR via heteroduplex rejection. Thus, the aim of this study was to investigate whether clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9)-mediated knock-in efficiency of human lactoferrin (hLF) knock-in vector in the bovine β-casein gene locus can be increased by suppressing DNA MMR-related genes (MSH2, MSH3, MSH6, MLH1, and PMS2) and overexpressing DNA double-strand break (DSB) repair-related genes (RAD51, CHK1, CHK2). Methods: Bovine mammary epithelial (MAC-T) cells were transfected with a knock-in vector, RAD51, CHK1, or CHK2 overexpression vector and CRISPR/sgRNA expression vector to target the bovine β-casein gene locus, followed by treatment of the cells with CdCl₂ for 24 hours. After 3 days of CdCl₂ treatment, the knock-in efficiency was confirmed by polymerase chain reaction (PCR). The mRNA expression levels of DNA MMR-related and DNA DSB repair-related genes were assessed by quantitative real-time PCR (RT-qPCR). Results: Treatment with CdCl₂ decreased the mRNA expression of RAD51 and MMRrelated genes but did not increase the knock-in efficiency in MAC-T cells. Also, the overexpression of DNA DSB repair-related genes in MAC-T cells did not significantly affect the mRNA expression of MMR-related genes and failed to increase the knock-in efficiency. Conclusion: Treatment with CdCl₂ inhibited the mRNA levels of RAD51 and DNA MMR-related genes in MAC-T cells. However, the function of MMR pathway in relation to HR may differ in various cell types or species.

• 원예과학기술지
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• 제30권6호
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• pp.734-742
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• 2012

본 연구는 배추의 유전자 기능분석을 위한 RNAi 유전자 침묵 기법과 T-DNA 삽입 기법을 비교하기 위해 수행하였다. 두 종류의 형질전환 계통이 이용되었으며 BrSAMS-knockout(KO) 계통은 T-DNA 삽입으로 한 개의 Brassica rapa S-adenosylmethionine synthetase(BrSAMS) 유전자가 기능을 상실한 계통이었으며 BrSAMS-knockdown(KD) 계통은 RNAi 방법을 통해 BrSAMS 유전자들의 발현이 억제된 계통이었다. KO 계통과 KD 계통의 microarray 분석 결과에서는 SAMS 유전자와 관련된 sterol, 자당, homogalacturonan 생합성 및 glutaredoxin-related protein, serine/threonine protein kinase, 그리고 gibberellin-responsive protein 유전자들의 발현 수준이 뚜렷한 차이를 보여 주었다. 그러나 KO 계통의 유전자 발현 양상은 하나의 BrSAMS 유전자가 기능을 상실하였음에도 불구하고 대조 계통과 비교하여 RNAi기법을 적용한 KD 계통에 비해 큰 차이를 보여주지 못했다. 또한 직접적으로 SAMS 유전자와 관련된 폴리아민과 에틸렌 합성 유전자들의 발현 변화도 KD 계통에서 더 잘 나타났다. 본 연구에서 microarray 결과를 이용한 KO 계통의 BrSAMS 기능분석은 배추과식물의 게놈 triplication 발생으로 인하여 다수로 존재하는 SAMS 유전자들 때문에 명확한 결론을 얻을 수 없었다. 결론적으로 배추와 같은 배수체 작물의 유전자 기능 분석은 RNAi silencing에 의한 유전자 knock-down 기법이 T-DNA 삽입에 의한 knock-out 기법보다 더욱 효율적인 것으로 나타났다.

• KSBB Journal
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• 제24권3호
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• pp.279-286
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• 2009

방선균은 다양한 생리활성 물질을 이차대사산물로 생산하는 산업적으로 매우 유용한 미생물이다. 이에 따라 많은 연구진들이 방선균에 대한 분자생물학적 연구와 산업적 이용에 대한 연구들을 수행하고 있다. 방선균 중에서도 S. avermitilis는 강력한 구충효과가 있는 avermectin을 생산하지만, 또한 포유동물 세포의 미토콘드리아에서 산화적 인산화반응을 억제하는 oligomycin도 함께 생성된다. 따라서 S. avermitilis에서 oligomycin의 생성을 제거시키기 위하여 oligomycin synthetase gene을 disruption 시키기 위한 연구를 수행하였다. 이를 위하여 S. avermitilis로부터 cloning 한 oligomycin synthetase gene (olmA5)의 중앙부분에 apramycin resistance gene을 끼워 넣어 integration vector로 구축한 후에 S. avermitilis의 chromosomal DNA와의 homologous recombination에 의하여 olmA5 gene의 disruption을 유도하였다. Disruption mutants (olmA5::apra)는 PCR을 통해 olmA5 gene의 위치에 apramycin resistance gene이 존재하는 것으로 확인하였고, 또한 HPLC 분석을 통해 oligomycin 생합성이 완전히 제거된 것임을 확인하였다. 그러나 disruption mutant (olmA5::apra)를 이용하여 avermectin 만을 생산할 수 있었으나, avermectin의 생산량에는 거의 변화가 없었다. 이러한 mutants는 산업적으로 avermectin을 생산하기 위한 균주 개량의 훌륭한 source가 될 수 있을 것이다.

• 대한의생명과학회지
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• 제26권2호
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• pp.114-119
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• 2020

Of the various types of mice used for genome editing, conditional knock-out (cKO) mice serve as an important model for studying the function of genes. cKO mice can be produced using loxP knock-in (KI) mice in which loxP sequences (34 bp) are inserted on both sides of a specific region in the target gene. These mice can be used as KO mice that do not express a gene at a desired time or under a desired condition by cross-breeding with various Cre Tg mice. Genome editing has been recently made easy by the use of third-generation gene scissors, the CRISPR-Cas9 system. However, very few laboratories can produce mice for genome editing. Here we present a more efficient method for producing loxP KI mice. This method involves the use of an HDR vector as the target vector and ssODN as the donor DNA in order to induce homologous recombination for producing loxP KI mice. On injecting 20 ng/µL of ssODN, it was observed that the target exon was deleted or loxP was inserted on only one side. However, on injecting 10 ng/µL of the target HDR vector, the insertion of loxP was observed on both sides of the target region. In the first PCR, seven mice were identified to be loxP KI mice. The accuracy of their gene sequences was confirmed through Sanger sequencing. It is expected that the loxP KI mice produced in this study will serve as an important tool for identifying the function of the target gene.

• Reproductive and Developmental Biology
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• 제38권2호
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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.

• 대한의생명과학회지
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• 제25권4호
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• pp.372-380
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• 2019

Mice with specific modified genes are useful means of studying development and disease. The CRISPR/Cas9 system is a very powerful and effective tool for generating genetically modified mice in a simple and fast manner. To generate human IgG light kappa constant knock-in mice, we tested by microinjection of a mixture of Cas9 protein, single-guide RNA and target homologous recombinant donor DNA into zygotes. We found that the injection of 10 ng/μL of Cas9 protein and crRNA/tracrRNA, rather than single guide RNA, induced the production of knock-in mice more effectively. Thus, our study provides valuable information that will help to improve the production of knock-in mice and contribute the successful generation of humanized Ab-producing mice in Korea.

• Molecules and Cells
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• 제37권10호
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• pp.734-741
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• 2014

Histone modifications on major transcription factor target genes are one of the major regulatory mechanisms controlling adipogenesis. Plant homeodomain finger 2 (PHF2) is a Jumonji domain-containing protein and is known to demethylate the histone H3K9, a repressive gene marker. To better understand the function of PHF2 in adipocyte differentiation, we constructed stable PHF2 knock-down cells by using the mouse pre-adipocyte cell line 3T3-L1. When induced with adipogenic media, PHF2 knock-down cells showed reduced lipid accumulation compared to control cells. Differential expression using a cDNA microarray revealed significant reduction of metabolic pathway genes in the PHF2 knock-down cell line after differentiation. The reduced expression of major transcription factors and adipokines was confirmed with reverse transcription- quantitative polymerase chain reaction and Western blotting. We further performed co-immunoprecipitation analysis of PHF2 with four major adipogenic transcription factors, and we found that CCATT/enhancer binding protein (C/EBP)${\alpha}$ and C/EBP${\delta}$ physically interact with PHF2. In addition, PHF2 binding to target gene promoters was confirmed with a chromatin immunoprecipitation experiment. Finally, histone H3K9 methylation markers on the PHF2-binding sequences were increased in PHF2 knock-down cells after differentiation. Together, these results demonstrate that PHF2 histone demethylase controls adipogenic gene expression during differentiation.

• Reproductive and Developmental Biology
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• 제32권4호
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• pp.229-235
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• 2008

Silencing of Dicer1 by siRNA did not inhibit development up to the blastocyst stage, but decreased expression of selected transcription factors, including Oct-4, Sox2 and Nanog, suggesting that Dicer1 gene expression is associated with differentiation processes at the blastocyst stage (Cui et al., 2007). In order to get insights into genes which may be linked with microRNA system, we compared gene expression profiles in Gapdh and Dicer1 siRNA-microinjected blastocysts using the Applied Biosystem microarray technology. Our data showed that 397 and 737 out of 16354 genes were up- and down-regulated, respectively, following siRNA microinjection (p<0.05), including 24 up- and 28 down-regulated transcription factors. Identification of genes that are preferentially expressed at particular Dicer1 knock down embryos provides insights into the complex gene regulatory networks that drive differentiation processes in embryos at blastocyst stage.

• Journal of Microbiology
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• 제44권3호
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• pp.320-326
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• 2006

Pasteurella multocida is an important veterinary and opportunistic human pathogen. In particular, strains of P. multocida serogroup D cause progressive atrophic rhinitis, and produce a potent, intracellular, mitogenic toxin known as P. multocida toxin (PMT), which is encoded by the toxA gene. To further investigate the toxigenic and pathogenic effects of PMT, a toxA-deleted mutant was developed by homologous gene recombination. When administrated to mice, the toxigenicity of the toxA mutant P. multocida was drastically reduced, suggesting that the PMT constributes the major part of the toxigenicity of P, multocida. Similar results were obtained in a subsequent experiment, while high mortalities were observed when toxA(+) P. multocida bacterial culture or culture Iysate were administrated. Mice immunized with toxA(-) P. multocida were not protected (none survived) following challenge with toxA(+) P. multocida or bacterial culture Iysate (toxin). These results suggest that the toxigenicity of P. multocida is mainly derived from PMT.

• Molecules and Cells
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• 제27권6호
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• pp.689-695
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• 2009

Chemically synthesized small interfering RNAs (siRNAs) can specifically knock-down expression of target genes via RNA interference (RNAi) pathway. To date, the length of synthetic siRNA duplex has been strictly maintained less than 30 bp, because an early study suggested that double-stranded RNAs (dsRNAs) longer than 30 bp could not trigger specific gene silencing due to the induction of non-specific antiviral interferon responses. Contrary to the current belief, here we show that synthetic dsRNA as long as 38 bp can result in specific target gene silencing without non-specific antiviral responses. Using this longer duplex structure, we have generated dsRNAs, which can simultaneously knock-down expression of two target genes (termed as dual-target siRNAs or dsiRNAs). Our results thus demonstrate the structural flexibility of gene silencing siRNAs, and provide a starting point to construct multifunctional RNA structures. The dsiRNAs could be utilized to develop a novel therapeutic gene silencing strategy against diseases with multiple gene alternations such as viral infection and cancer.