• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2002년도 춘계학술발표대회 발표논문초록집
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• pp.87-87
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• 2002

Previously, we observed high level expression of goat β-casein/genomic hGH fusion gene in mammary gland of transgenic mice. To develop an expression vector to make a human granulocyte-colony stimulating factor (hG-CSF) protein efficiently produced in milk of transgenic animals, we designed a new bicistronic vector using the goat β-casein/genomic hGH fusion gene as regulation sequences for expression and internal ribosome entry site (IRES) as a mediator for second gene expression. This vector was constructed by insertion of encephalomyocarditis virus (EMCV) IRES-dependent second gene region coupled with hG-CSF cDNA into 3' untranslated region of an intact hGH gene. By microinjcetion, four transgenic mice were generated and three of them transmitted the bicistronic vector to their progeny. (omitted)

• 한국발생생물학회지:발생과생식
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• 제17권1호
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• pp.1-8
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• 2013

Osteoprotegerin (OPG) is a secreted glycoprotein that regulates bone resorption by inhibiting differentiation and activation of osteoclast, thereby potentially useful for the treatment of many bone diseases associated with increased bone loss. In this study, we designed a novel cDNA expression cassette by modifying the potent and mammary gland-specific goat ${\beta}$-casein/hGH hybrid gene construct and examined human OPG (hOPG) cDNA expression in transgenic mice. Six transgenic mice all successfully expressed hOPG in their milk at the level of 0.06-2,000 ${\mu}g/ml$. An estimated molecular weight of the milk hOPG was 55 kDa in SDS-PAGE, which is the same as a naturally glycosylated monomer. This hOPG expression was highly specific to the mammary glands of transgenic mice. hOPG mRNA was not detected in any organs analyzed except mammary gland. Functional integrity of milk hOPG was evaluated by TRAP (tartrate-resistant acid phosphatase) activity assay in bone marrow cell cultures. OPG ligand (OPG-L) treatment increased TRAP activity by two fold but it was completely abolished by co-treatment with transgenic milk containing hOPG. Taken together, our novel cDNA expression cassette could direct an efficient expression of biologically active hOPG, a potential candidate pharmaceutical for bone diseases, only in the mammary gland of transgenic mice.

• 한국발생생물학회지:발생과생식
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• 제14권1호
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• pp.13-19
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• 2010

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 $237{\mu}g/m{\ell}$ and $8,990{\mu}g/m{\ell}$, 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 ($91{\mu}g/m{\ell}$) 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.

• Reproductive and Developmental Biology
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• 제40권1호
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• pp.7-13
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• 2016

Effectiveness of transgene transfer into genome is crucially concerned in mass production of the bio-pharmaceuticals using genetically modified transgenic animals as a bioreactor. Recently, the mammary gland has been considered as a potential bioreactor for the mass production of the bio-pharmaceuticals, which appears to be capable of appropriate post-translational modifications of recombinant proteins. The mammary gland tissue specific vector system may be helpful in solving serious physiological disturbance problems which have been a major obstacle in successful production of transgenic animals. In this study, to minimize physiological disturbance caused by constitutive over-expression of the exogenous gene, we constructed new retrovirus vector system designed for mammary gland-specific expression of the hEPO gene. Using piggyBac vector system, we designed to express hEPO gene under the control of mammary gland tissue specific and lactogenic hormonal inducible goat ${\beta}$-casein or mouse Whey Acidic Protein (mWAP) promoter. Inducible expression of the hEPO gene was confirmed using RT-PCR and ELISA in the mouse mammary gland cells treated with lactogenic hormone. We expect the vector system may optimize production efficiency of transgenic animal and reduce the risk of global expression of transgene.

• Asian-Australasian Journal of Animal Sciences
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• 제22권2호
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• pp.168-173
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• 2009

Human follicle-stimulating hormone (hFSH) is a pituitary glycoprotein that regulates follicular development and ovulation. Clinically, hFSH has been used to induce follicular growth in infertile women. The hormone is composed of heterodimers, including a common ${\alpha}$ subunit among the gonadotropin family and a hormone-specific ${\beta}$ subunit. Since assembly of the heterodimer is a rate-limiting step in the production of functional hFSH, transgenic clone cows carrying a single-chain hFSH transgene may efficiently produce functional hormone. Genes encoding the ${\alpha}$ and ${\beta}$ subunits of hFSH were linked using the C-terminal peptide sequence from the ${\beta}$ subunit of human chorionic gonadotropin. Bovine fetal fibroblasts were transfected with the gene construct, including the goat ${\beta}$-casein promoter and a single-chain hFSH coding sequence. Transfected fibroblasts were transferred into enucleated oocytes, and individual nuclear transfer (NT) embryos developed to the blastocyst stage were analyzed for the transgene by polymerase chain reaction. Seventy eight blastocysts (30.8%) were developed from 259 reconstructed embryos. Among these blastocysts, the hFSH gene was detected in 70.8% (34/48) of the embryos. Subsequent transfer of hFSH-transgenic clone embryos to 31 recipients results in 11 (35.5%) early pregnancies. However, all fetuses were lost before reaching day 180 of gestation. The results from this study demonstrated that bovine NT embryos carrying single-chain hFSH could be produced, and further extensive studies in which NT embryos are transferred to more recipients may give rise to single chain hFSH-transgenic cows for biomedical applications.

• 한국식품과학회지
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• 제46권2호
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• pp.121-126
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• 2014

산양유는 단백질 구성과 개별 지방산의 구성이 모유에 가깝기 때문에 우유에 비하여 소화율이 높다. 특히 casein 단백질은 모유의 단백질과 분자구조가 흡사하여 높은 소화 흡수력을 보이므로 설사 또는 복통 등과 같은 소화장애를 일으키지 않는다. 뿐만 아니라 지방산 함량에 있어서도 우유보다 중간사슬지방산이 우유보다 2배정도 많이 들어있어 소화 흡수 시 분해되는 속도가 빠르다. 산양유에는 질 좋은 수용성 유청단백질, 비타민 및 미네랄이 다량 함유되어 있고, 특히 비타민 A, 콜린, 이노시톨의 함량이 높고 흡수율도 양호한 편이다. 산양유에 포함된 유당과 올리고당은 우유보다 모유에 더 가까운 효과를 기대할 수 있다. 유당은 장 내 산성을 증가시켜 유익한 박테리아의 증식을 돕는 한편 장내에서의 칼슘의 흡수를 도와주거나 인(P)과 마그네슘(Mg)의 흡수를 촉진시키는 역할을 담당한다. 또한 올리고당류는 장내유산균의 생육을 돕는 prebiotic 효과를 발휘하여 장내 유익균이 정착하는데 도움을 준다는 실험결과로 판단할 때 실제로 유아에게 정장작용을 기대할 수 있다. 그리고 산양유에 다량 함유된 미네랄 성분 또한 소화 및 흡수를 촉진시키는 역할을 하기 때문에 산양유는 우유 섭취 시 유당불 내증을 호소하던 소비자들도 어렵지 않게 섭취할 수 있다. 이처럼 산양유가 여러 가지 측면에서 우유보다 우수한 식품학적 생리적 특성을 가지고 있음에도 불구하고 국내 산양유가공 산업은 거의 불모지라 해도 과언이 아니다. 게다가 산양유의 정확한 영양학적 가치가 일반 소비자들에게 전달되지 못하고 있는 실정이다. 따라서 우유보다 그 성분과 조성이 모유와 흡사한 산양유의 식품학적 특성, 안전성 및 기능성에 대한 체계적이며 과학적 연구가 시급히 활성화되어야 할 것이다.