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- 저자 : Koo Okjae (검색결과 3 건)
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Hossein, Mohammad Shamim,Kim, Min Kyu,Jang, Goo,Oh, Hyun Joo,Koo, OkJae,Kim, Jeong Joo,Kang, Sung Keun,Lee, Byeong Chun,Hwang, Woo Suk JOHN WILEY & SONS LTD 2007 MOLECULAR REPRODUCTION AND DEVELOPMENT Vol.74 No.9
<P>Various thiol compounds are known to improve cytoplasmic and/or nuclear maturation of oocytes in vitro. The present study examined the effects of two thiol compounds, cysteine (0.1, 0.5, and 1.0 mM) and cysteamine (50, 100, and 200 µM), on cytoplasmic and nuclear maturation of canine oocytes. Oocytes collected from different reproductive stages were cultured in TCM-199 supplemented with 10% fetal bovine serum, 2.2 mg/ml sodium carbonate, 2.0 µg/ml estrogen, 0.5 µg/ml FSH, 0.03 IU/ml hCG, and 1% penicillin–streptomycin solution for 72 h. Data were analyzed by two-way ANOVA after arcscine transformation and protected by Bonferroni post hoc test. The effects of cysteine and cysteamine on canine IVM were varied depending on the reproductive stage of oocyte donor bitches. In the follicular stage, significantly more oocytes reached the metaphase II (M II) stage when cultured with 0.5 or 1.0 mM cysteine (16.7% and 16.9%, respectively) compared to the control (6.2%). In the follicular stage, cysteamine increased oocyte maturation rate upto the M II stage (15.1% to 17.0%) compared to the control (4.4%). Both the 0.5 mM cysteine and 100 µM cysteamine, alone or together, increased the intracellular GSH level of canine oocytes compared to the control. Irrespective of reproductive stage, no further beneficial effects on nuclear or cytoplasmic maturation were observed when 0.5 mM cysteine and 100 µM cysteamine were supplemented together. In conclusion, addition of 0.5 mM cysteine and 100 µM cysteamine to the maturation medium improved IVM of canine oocytes. Mol. Reprod. Dev. 74: 1213–1220, 2007. © 2007 Wiley-Liss, Inc.</P>
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JoonHo Moon,SuJin Kim,HeeJung Park,JungTaek Kang,SolJi Park,OkJae Koo,Begona Roibas da Torre,Islam M. Saadeldin,ByeongChun Lee,Goo Jang 한국동물생명공학회(구 한국동물번식학회) 2011 발생공학 국제심포지엄 및 학술대회 Vol.2011 No.1
The necessity of conditional gene expression in pigs for transgenic models is raised. Thus, in this study, Cre-loxP conditional expression in porcine fetal fibroblasts was investigated and the transformed fibroblasts were reprogrammed in enucleated oocytes for further early embryonic development. Fetal fibroblasts from miniature pigs were used for transfection with pCALNL-DsRed including floxed neomycin resistant gene and selected with 750 ug/mL neomycin for two weeks. The transfected cells did not express DsRed under fluorescence microscope. After transient transfection of plasmid DNA expressing Cre, the fibroblasts began to express DsRed. The cells expressing Ds- Red were employed into somatic cell nuclear transfer (SCNT). A total of 121 oocytes were used for SCNT and 76 cloned embryos (62.8%)were cleaved. Six blastocysts were grown up after SCNT and expressed DsRed. Deletion of floxed neomycin resistant gene was confirmed by RT-PCR in cloned blastocysts. Taken together, this study demonstrated that Cre-loxP recombination in miniature pig fibroblasts were successfully worked and those sequential transformed cells were developed into pre-implantation stage via SCNT.
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Lee Myoung Hui,Lee Jiyoung,Choi Seung A,Kim Ye-Sol,Koo Okjae,Choi Seung Hee,Ahn Woo Seok,Jie Eun Yee,Kim Suk Weon 한국식물생명공학회 2020 Plant biotechnology reports Vol.14 No.6
Nowadays, genome editing in plants has become much easier thanks to the recently developed clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 9 (CRISPR–Cas9) nuclease system. However, to combine protoplast technology with the CRISPR–Cas9 system in plants, a stable and an efcient foreign DNA delivery system is essential for gene editing. In the present study, we developed an electro-transfection system for CRISPR–Cas9 ribonucleoprotein (RNP) delivery to cabbage protoplasts. Under 1000 V treatment, the frequency of initial cell division and total number of cell colonies formed were 47.7±2.5% and 52±7.5%, respectively. The total number of cell colonies formed following 1000 V treatment was 1.4 times higher than that following polyethylene glycol (PEG) treatment. However, the frequency of initial cell division and total number of cell colonies formed from protoplasts decreased with increasing voltage. Cy3–Cas9 protein delivery into the nucleus was confrmed through both electro-transfection and PEG-mediated transfection using confocal laser scanning microscopy. The frequency of insertions and deletions in the synthesized guide RNA of phytoene desaturase 1 was the highest at 3.4% following electro-transfection at 1000 V with a pulse width of 20 ms and only 1.8% following PEG-mediated transfection. These results indicate that electro-transfection is more efcient in RNP delivery to protoplast than PEG-mediated transfection in cabbage for PDS1 sgRNA delivery. Therefore, the electro-transfection system developed in the present study presents the possibility it could be used for DNA-free genome editing of other crops.
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