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Oh Keon-Bong,Lee Chul-Sang 한국발생생물학회 2010 발생과 생식 Vol.14 No.1
We developed a novel dicistronic system for the expression of target cDNA sequences in the milk of transgenic animals using goat beta-casein/hGH fusion construct, pGbc5.5hGH (Lee, 2006) and internal ribosome entry site (IRES) sequences of encephalomyocarditis virus (EMCV). Granulocyte colony-stimulating factor (hG-CSF) cDNA was linked to 3' untranslated region of hGH gene in the pGbc5.5hGH via EMCV IRES sequences. Transgenic mice were generated by microinjection and transgene expression was examined in the milk and mammary gland of transgenic mice at 10 days of lactation. Northern blot analysis showed that hGH gene and hG-CSF cDNA were transcribed as a single dicistronic mRNA. The hG-CSF and hGH proteins were independently translated from the dicistronic mRNA and secreted into the milk of transgenic mice. The highest concentration of hG-CSF and hGH in the milk of transgenic mice were and , respectively. In contrast, another hG-CSF expression cassette, in which hG-CSF genomic sequences were inserted into a commercial milk-specific expression vector (pBC1), generated a lower level () of hG-CSF expression in the milk of transgenic mice. These results demonstrated that the novel pGbc5.5hGH-based dicistronic construct could be useful for an efficient cDNA expression in the milk of transgenic animals.
Growth Factor Supplementation in Culture Medium Leads to Active Proliferation of Porcine Fibroblast
Keon Bong Oh,Bella Kim,Nayoung Ko,Seong-soo Hwang,Dong-Hoon Kim,Gi-Sun Im,Byoung-Chul Yang,Jae-Seok Woo,Zae Young Ryoo,Jin-Ki Park 한국동물생명공학회(구 한국동물번식학회) 2011 Reproductive & developmental biology Vol.35 No.2
Generation of Heterozygous Gal KO Fibroblast Expressing Efficiently MCP at GalT Locus
Keon Bong Oh,Nayouna Ko,Bella Kim,Seong-soo Hwang,Jeong Woong Lee,Dong-Hoon Kim,Gi-Sun Im,Byoung-Chul Yang,Man-Jong Kang,Choon-Keun Park,Jin-Ki Park 한국동물생명공학회(구 한국동물번식학회) 2011 Reproductive & developmental biology Vol.35 No.2
Growth Factor Supplementation in Culture Medium Leads to Active Proliferation of Porcine Fibroblast
Keon Bong Oh,Bella Kim,Nayoung Ko,Seong-soo Hwang,Dong-Hoon Kim,Gi-Sun Im,Byoung-Chul Yang,Jae-Seok Woo,Zae Young Ryoo,Jin-Ki Park 한국동물번식학회 2011 Reproductive & Developmental Biology(Supplement) Vol.35 No.2s
Generation of Transgenic Pigs Carrying a Construct for Endothelial Specific Expression of CD73
Keon Bong Oh,Bella Kim,Nayoung Ko,Dae-Jin Kwon,Dong-Hoon Kim,Gi-Sun Im,Jin-Ki Park,Seongsoo Hwang 한국동물생명공학회(구 한국동물번식학회) 2011 발생공학 국제심포지엄 및 학술대회 Vol.2011 No.1
Despite of the absence of hyperacute rejection and acute humoral xenograft rejection, the organ graft of the a1,3-galactosyltransferase (GalT) gene knockouted (KO) and complement regulatory protein (CRP) expressing pig into a nonhuman primate is rejected by development of a thrombotic microangiopathy and/or a consumptive coagulopathy. Thus further introduction of genes to overcome the coagulation incompatibilities between pig and primate under GalT KO/CRP genetic background has been strongly suggested. CD73 (ecto-5'-nucelotidase) is an enzyme attached via a glycosyl phosphoinositol anchor to the extracellular membrane of endothelial cells, which catalyses the hydrolysis of adenosine triphosphate to adenosine. Loss of activity of CD73 results in activation and aggregation of platelets by a reduced capacity to convert nucleotides to adenosine. In previous study, we reported generation of GalT KO fibroblasts concurrently expressing membrane cofactor protein and produced cloned pigs by nuclear transfer of the fibroblast cells (1). In this study, we constructed a vector for expression of human CD73 under control of promoter of pig Icam2 gene expressed specifically at endothelial cells. This vector was introduced into porcine fibroblasts using the nucleofection technology, by which we had forty three fibroblasts clones carrying pIcam2- CD73 vector. Somatic cell nuclear transfer resulted in generation of two transgenic piglets survived.
Generation of Heterozygous Gal KO Fibroblast Expressing Efficiently MCP at GalT Locus
Keon Bong Oh,Nayouna Ko,Bella Kim,Seong-soo Hwang,Jeong Woong Lee,Dong-Hoon Kim,Gi-Sun Im,Byoung-Chul Yang,Man-Jong Kang,Choon-Keun Park,Jin-Ki Park 한국동물번식학회 2011 Reproductive & Developmental Biology(Supplement) Vol.35 No.2s
Oh Keon-Bong,Lee Chul-Sang 한국발생생물학회 2009 발생과 생식 Vol.13 No.3
In an attempt to simultaneously produce two human proteins, hGH and hG-CSF, in the milk of transgenic mice, we constructed goat -casein-directed hGH and hG-CSF expression cassettes individually and generated transgenic mice by co-injecting them into mouse zygotes. Out of 33 transgenic mice, 29 were identified as double transgenic harboring both transgenes on their genome. All analyzed double transgenic females secreted both hGH and hG-CSF in their milks. Concentrations ranged from 2.1 to for hGH and from 0.04 to for hG-CSF. hG-CSF level was much lower than hGH level but very similar to that of single hG-CSF mice, which were introduced with hG-CSF cassette alone. In order to address the causes of concentration difference between hGH and hG-CSF in milk, we examined mRNA level of hGH and hG-CSF in the mammary glands of double transgenic mice and tissue specificity of hG-CSF mRNA expression in both double and single transgenic mice. Likewise protein levels in milk, hGH mRNA level was much higher than hG-CSF mRNA, and hG-CSF mRNA expression was definitely specific to the mammary glands of both double and single transgenic mice. These results demonstrated that two transgenes have distinct transcriptional potentials without interaction each other in double transgenic mice although two transgenes co-integrated into same genomic sites and their expressions were directed by the same goat -casein promoter. Therefore goat -casein promoter is very useful for the multiple production of human proteins in the milk of transgenic animals.