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소 β-casein 유전자 영역에서 소 Insulin-like Growth Factor 1을 생산하기 위한 Knock-in Vector
김상영,박다솜,김세은,구덕본,강만종,Kim, Sang Young,Park, Da Som,Kim, Se Eun,Koo, Deog-Bon,Kang, Man-Jong 한국동물번식학회 2017 Reproductive & developmental biology Vol.41 No.3
The production of therapeutic protein from transgenic domestic animal is the major technology of biotechnology. Insulin-like growth factor-1 (IGF-1) is known to play an important role in the growth of the animal. The objective of this study is construction of knock-in vector that bovine IGF-1 gene is inserted into the exon 7 locus of ${\beta}$-casein gene and expressed using the gene regulatory DNA sequence of bovine ${\beta}$-casein gene. The knock-in vector consists of 5' arm region (1.02 kb), bIGF-1 cDNA, CMV-EGFP, and 3' arm region (1.81 kb). To express bIGF-1 gene as transgene, the F2A sequence was fused to the 5' terminal of bIGF-1 gene and inserted into exon 7 of the ${\beta}$-casein gene. As a result, the knock-in vector is confirmed that the amino acids are synthesized without termination from the ${\beta}$-casein exon 7 region to the bIGF-1 gene by DNA sequence. These knock-in vectors may help to create transgenic dairy cattle expressing bovine bIGF-1 protein in the mammary gland via the expression system of the bovine ${\beta}$-casein gene.
김민지 ․ 구덕본 대구대학교 산업기술연구소 2018 産業技術硏究 Vol.29 No.2
Production of transgenic animals are being used in various fields such as commercial application, human diseases model and drug discovery. One of these animals production method of transgenic animal is direct transfer by pronuclear microinjection. However, the production efficiency of transgenic animals using the pronuclear microinjection is remarkably low. In addition, appropriate conditions such as improving the embryo developmental competence and high quality of blastocysts for the production of transgenic embryos are being required, but studies for these process have not been reported. Therefore, this study investigated the embryonic developmental competence and quality of blastocysts after pronuclear microinjection by using cytochalasin B (CB) or/and melatonin. Cytochalasin B (CB) is important to play role in microinjection by making smoother of the zygote cytoplasm, as well as melatonin is known to improve the embryonic developmental competence and embryos quality. First, we performed the microinjection using injection medium with 10 µg/ml CB and cultured the microinjected zygotes in 0.1 µM melatonin treated within culture medium. Next, we confirmed the DNA damage of microinjected blastocysts using immunofluorescence staining with RAD51 (DNA repairs detection protein) and H2AX139ph (DNA damage detection protein). CB treated group significantly increased blastocyst development rate compared with CB non-treated group (P < 0.05; 25.0 ± 7.2% vs. 17.0 ± 1.3%). After microinjection under the CB treatment, blastocyst development rate and formation of expanded blastocysts were higher in melatonin treated group than those of melatonin non-treated group (29.7 ± 8.3% vs. 24.4 ± 7.7%). In addition, melatonin treated group increased the numbers of RAD51 positive cells compared with non-treated group (P < 0.05). Taken together, these results suggest that CB and melatonin treatment have positive effects for improving the production efficiency of transgenic embryos.