초록 ▼

Ⅳ. 연구개발결과
본 과제에서는 젖소 β-casein 유전자 엑손 7위치의 상류 또는 하류 유전자와 엔도스타틴 유전자를 동정하여 전체 염기서열을 결정하였다. 또한 이들 유전자를 이용하여 DT-A_cEndo_neo KI vector, DT-A_tEndo_neo KI vector I, DT-A_tEndo_neo KI vector II 및 DT-A_tEndo_EGFP KI vector II 벡터를 제작하였다. 엑손 7을 절단할 수 있는 TALEN을 합성하여 T7 endonuclease assay와 reporter gene assay

Ⅳ. 연구개발결과
본 과제에서는 젖소 β-casein 유전자 엑손 7위치의 상류 또는 하류 유전자와 엔도스타틴 유전자를 동정하여 전체 염기서열을 결정하였다. 또한 이들 유전자를 이용하여 DT-A_cEndo_neo KI vector, DT-A_tEndo_neo KI vector I, DT-A_tEndo_neo KI vector II 및 DT-A_tEndo_EGFP KI vector II 벡터를 제작하였다. 엑손 7을 절단할 수 있는 TALEN을 합성하여 T7 endonuclease assay와 reporter gene assay을 실시하여 validation을 실시하였다. 그 결과 TALEN2에서 reporter 유전자를이용하여 RFP-GFP 발현을 확인한 결과 11.9%의 활성을 나타내었다. 또한 T7 endonuclease assay에 의해서도 indel 현상이 유도됨을 확인하였다. 이들 TALEN과 knock-in 벡터를 젖소 체세포에 도입한 결과 약 2.4 - 4.8%의 유전자 적중 효율을 얻을 수 있었다. 또한 소 전핵기수정란에 TALEN과 knock-in 벡터을 co-injection 한 후 배반포기까지 자란 수정란을 분석한 결과 33%의 유전자 적중 효율을 나타냄을 확인하였다. 따라서 본 과제에서 개발된 시스템은 수정란에 TALEN과 knock-in 벡터를 주입하여 형질전환 젖소를 생산하는데 이용될 수 있을 것으로 생각된다.

Abstract ▼

Many transgenic domestic animals have been developed to produce therapeutic proteins in the mammary gland, and this approach is one of the most important methods for agricultural and biomedical applications. However, expression and secretion of a protein varies because transgenes are integrated at r

Many transgenic domestic animals have been developed to produce therapeutic proteins in the mammary gland, and this approach is one of the most important methods for agricultural and biomedical applications. However, expression and secretion of a protein varies because transgenes are integrated at random sites in the genome. In addition, the distal enhancers are very important for transcriptional gene regulation and tissue-specific gene expression. Endostatin is 20-KDa C-terminal fragment derived from type XVIII collagen and an endogenous inhibitor of tumor growth by inhibition of angiogenesis. Generating transgenic dairy cattle to produce endostatin in milk may help in the treatment cancer therapy. The objective of this study was to develop a knock-in system for expression of endostatin in the bovine β-casein gene locus. The knock-in vector consists of 5' arm region and 3'arm region, and neomycin resistance gene (neor)as a positive selection marker gene. The F2Asequence was fused to the 5' terminal of endostatin gene and inserted into exon7 of the β-casein gene. We constructed 4 kind of knock-in vector; DT-A_cEndo_neo KI, DT-A_tEndo_neo KI vector I, DT-A.tEndo_neo KI vector II and DT-A.tEndo-EGFP KI vector II. We were co-transfected DT-A_cEndo_neo KI, DT-A_tEndo_neo KI vector I and DT-A.tEndo_neo KI vector II and TALEN into the bovine somatic cells by electroporation and produced knock-in somatic cells. Our knock-in somatic cells may help to create cloned embryos for development of transgenic dairy cattle expressing human endostatin protein in the mammary gland via the expression system of the bovine β-casein gene. 3 out of 65 colonies in the DT-A_cEndo_neo KI vector were validated as positive by 2^ndPCR.In addition, in the DT-A_tEndo_neo KI vector I and DT-A.tEndo_neo KI vector II, 5 out of 210 and 3 out of 62 colonies were identified as positive by 2^ndPCR, respectively. However, after co-injection of DT-A.tEndo-EGFP KI vector II and TALEN into one cell embryo, we was obtained knock-in blastocyst (33.3%). Our knock-in system may help to create knock-in fibroblasts and embryos for development of transgenic dairy cattle expressing human endostatin protein in the mammary gland via the expression system of the bovine β-casein gene.