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Kim, Teoan,Lee, Young Man,Lee, Hoon Taek,Heo, Young Tae,Yom, Heng-Cherl,Kwon, Mo Sun,Koo, Bon Chul,Whang, Key,Roh, Kwang Soo Asian Australasian Association of Animal Productio 2001 Animal Bioscience Vol.14 No.2
Despite the high potency of the retrovirus vector system in gene transfer, one of the main drawbacks of has been difficulty in preparing highly concentrated virus stock. Numerous efforts to boost the virus titer have ended in unsatisfactory results mainly due to fragile property of retrovirus envelope protein. In this study, to overcome this problem, we constructed our own retrovirus vector system producing vector viruses encapsulated with VSV-G (vesicular stomatitis virus G glycoprotein). Concentration process of the virus stock by ultracentrifuge did not sacrifice the virus infectivity, resulting in more than 108 to 109 CFU (colony forming unit) per ml on most of the target cell lines tested. Application of this high-titer retrovirus vector system was tested on chicken embryos. Injection of virus stock beneath the blastoderms of pre-incubated fertilized eggs resulted in chick embryos expressing E. coli LacZ gene with 100% efficiency. Therefore, our results suggest that it is possible to transfer the foreign gene into chicken embryo using our high-titer retrovirus vector.
Lentivirus-mediated Gene Transfer to Bovine Embryos
Young Mi Kim,Mo Sun Kwon,Bon Chul Koo,Teoan Kim,Heng-Cherl Yom,Dae Hwan Ko 한국동물생명공학회(구 한국동물번식학회) 2008 Reproductive & developmental biology Vol.32 No.1
Pronuclear DNA microinjection has been the most universal method in transgenic animal production but its success rate of transgenesis in mammals are extremely low. To address this long-standing problem, we used retrovirus- and lentivirus-based vectors carrying the enhanced green fluorescent protein (EGFP) gene under the control of ubiquitously active cytomegalovirus (CMV) promoter to deliver transgenes to bovine embryos. The rate of transgenesis was evaluated by counting EGFP positive blastocysts after injection of concentrated virus stock into the perivitelline space of the bovine oocytes in metaphase II. Among two different types of lentivirus vectors derived from FIV (feline immunodeficiency virus) and HIV (human immunodeficiency virus), the former scored the higher gene transfer efficiency; almost 100% of the blastocysts developed from the oocytes infected with FIV-based vector were EGFP positive. As for the vectors derived Com HIV lentivirus, the transgenesis rate of the blastocysts was reduced to 39%.
Cho, Seong-Keun,Kim, Jae-Hwan,Park, Jong-Yi,Choi, Yun-Jung,Bang, Jae-Il,Hwang, Kyu-Chan,Cho, Eun-Jeong,Sohn, Sea-Hwan,Uhm, Sang Jun,Koo, Deog-Bon,Lee, Kyung-Kwang,Kim, Teoan,Kim, Jin-Hoi Wiley-Liss,Inc 2007 Developmental dynamics Vol.236 No.12
<P>Somatic cell nuclear transfer (scNT) is a useful way to create cloned animals. However, scNT clones exhibit high levels of phenotypic instability. This instability may be due to epigenetic reprogramming and/or genomic damage in the donor cells. To test this, we produced transgenic pig fibroblasts harboring the truncated human thrombopoietin (hTPO) gene and used them as donor cells in scNT to produce first-generation (G1) cloned piglets. In this study, 2,818 scNT embryos were transferred to 11 recipients and five G1 piglets were obtained. Among them, a clone had a dimorphic facial appearance with severe hypertelorism and a broad prominent nasal bridge. The other clones looked normal. Second-generation (G2) scNT piglets were then produced using ear cells from a G1 piglet that had an abnormal nose phenotype. We reasoned that, if the phenotypic abnormality of the G1 clone was not present in the G2 and third-generation (G3) clones, or was absent in the G2 clones but reappeared in the G3 clones, the phenotypic instability of the G1 clone could be attributed to faulty epigenetic reprogramming rather than to inherent/accidental genomic damage to the donor cells. Blastocyst rates, cell numbers in blastocyst, pregnancy rates, term placenta weight and ponderal index, and birth weight between G1 and G2 clones did not differ, but were significantly (P < 0.05) lower than control age- and sex-matched piglets. Next, we analyzed global methylation changes during development of the preimplantation embryos reconstructed by donor cells used for the production of G1 and G2 clones and could not find any significant differences in the methylation patterns between G1 and G2 clones. Indeed, we failed to detect the phenotypic abnormality in the G2 and G3 clones. Thus, the phenotypic abnormality of the G1 clone is likely to be due to epigenetic dysregulation. Additional observations then suggested that expression of the hTPO gene in the transgenic clones did not appear to be the cause of the phenotypic abnormality in the G1 clones and that the abnormality was acquired by only a few of the G1 clone's cells during its gestational development. Developmental Dynamics 236:3369–3382, 2007. © 2007 Wiley-Liss, Inc.</P>
Generation of red fluorescent protein transgenic dogs
Hong, So Gun,Kim, Min Kyu,Jang, Goo,Oh, Hyun Ju,Park, Jung Eun,Kang, Jung Taek,Koo, Ok Jae,Kim, Teoan,Kwon, Mo Sun,Koo, Bon Chul,Ra, Jeong Chan,Kim, Dae Yong,Ko, CheMyong,Lee, Byeong Chun Wiley Subscription Services, Inc., A Wiley Company 2009 Genesis Vol.47 No.5
<P>Dogs (Canis familiaris) share many common genetic diseases with humans and development of disease models using a transgenic approach has long been awaited. However, due to the technical difficulty in obtaining fertilizable eggs and the unavailability of embryonic stem cells, no transgenic dog has been generated. Canine fetal fibroblasts were stably transfected with a red fluorescent protein (RFP) gene-expressing construct using retrovirus gene delivery method. Somatic cell nuclear transfer was then employed to replace the nucleus of an oocyte with the nucleus of the RFP-fibroblasts. Using this approach, we produced the first generation of transgenic dogs with four female and two male expressing RFP. genesis 47:314–322, 2009. © 2009 Wiley-Liss, Inc.</P>
Jeong, Yu-Jeong,Cui, Xiang-Shun,Kim, Bong-Ki,Kim, Il Hwa,Kim, Teoan,Chung, Yon-Bok,Kim, Nam-Hyung Cambridge University Press 2005 Zygote Vol.13 No.1
<P>The objective of this study was to determine developmental pattern, total cell number, apoptosis and apoptosis-related gene expression in haploid and diploid embryos following parthenogenetic activation. <I>In vitro</I>-matured porcine oocytes were activated by electrical pulses and cultured in the absence or presence of cytochalasin B for 3 h. Zygotes with two polar bodies (haploid) and one polar body (diploid) were carefully selected and were further cultured in NCSU 23 medium containing 0.4% bovine serum albumin (BSA) for 7 days. The percentage of development to blastocyst stage was higher (<I>p</I><0.01) in the diploid than in the haploid parthenotes. In haploid blastocysts, average total cell number was significantly reduced (<I>p</I><0.05) and apoptosis was increased at day 7. The relative abundance of Bcl-xL and Bak mRNA in the diploid blastocysts was similar to that of <I>in vivo</I>-fertilized embryos. However, Bcl-xL was significantly decreased, and Bak mRNA was significantly increased (<I>p</I><0.05) in haploid parthenotes compared with the diploid parthenotes. These results suggest that the haploid state affects apoptosis-related gene expression which results in increased apoptosis and decreased developmental competence of haploid parthenotes.</P>