Earticle

The recombinant production of therapeutic proteins from transgenic animal is the major technology of animal biotechnology and is one of the biggest challenges in an efficient bioreactor in the pharmaceutical industry. The useful gene delivery into cells or animals is an important way to express the target gene in mammary glands. Tissue plasminogen activator (tPA) is known to play an important role in the major enzyme responsible for blood clots breakdown. Because it works on the clotting system, tPA is used in clinical medicine to treat thrombolysis, such as pulmonary embolism, myocardial infraction and stroke. The objective of this study is a production of recombinant human tPA protein in the milk of porcine. For this study, we construct knock-in vector that human tPA gene is inserted into the exon 1 locus of porcine whey acid protein (pWAP) gene knock-out somatic cell by homologous recombination. Finally, establishment of tPA knock-in cell lines were confirmed by genomic PCR analysis and identification of karyotype. As a result, we established cell lines of pWAP knock-out expressing human tPA gene from porcine fibroblasts to apply somatic cell nuclear transfer (SCNT). Further study will be performed for the production of transgenic pigs.

본 연구는 돼지 B-casein 유전자 위치에서 EGFP가 발현될 수 있는 knock-in 벡터를 구축하기 위하여 실시되었다. 돼지의 B-casein 유전자를 이용하여 knock-in 벡터를 구축하기 위해 돼지의 태아 섬유아세포로부터 B-casein 유전자를 동정하였고 EGFP, SV4O polyA signal을 동정하였다. Knock-in 벡터는 5' 상동 영역 약 5 kb와 3' 상동 영역 약 2.7 kb로 구성되어있으며, positive selection marker로 neor 유전자를, negative selection marker로 DT-A 유전자를 사용하였다. 구축된 knock-in 벡터로부터 EGFP의 발현을 확인하기 위하여 생쥐 유선 세포인 HC11 세포에 knock-in 벡터를 도입하였다. 그 결과 EGFP의 발현을 HC11 세포에서 확인하였다. 이와 같은 결과로서 이 block-in 벡터는 knock-in 형질전환 돼지를 생산하는데 사용될 수 있을 것으로 생각된다.

This study was carried out to develop knock-in vector for EGFP (enhanced green fluorescent protein) expression in porcine B-casein locus. For construction of knock-in vector using porcine B-casein gene, we cloned the B-casein genome DNA from porcine fetal fibroblast cells, EGFP and SV40 polyA signal using PCR. The knock-in vectors consisted of a 5-kb fragment as the 5' recombination arm and a 2.7-kb fragment as the 3' recombination arm. We used the neomycin resistance gene (neor) as a positive selectable marker and the diphtheria toxin A (DT-A) gene as a negative selectable marker. To demonstrate EGFP expression from knock-in vector, we are transfected knock-in vector that has EGFP gene in murine mammary epithelial cell line HC11 cells with pSV2 neo plasmid. The EGFP expression was detected in HC11 cells transfected knock-in vector. This result demonstrates that this knock-in vector may be used for the development of knock-in transgenic pig.

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 β-casein gene and expressed using the gene regulatory DNA sequence of bovine β-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 β-casein gene. As a result, the knock-in vector is confirmed that the amino acids are synthesized without termination from the β-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 β-casein gene.

The production of therapeutic protein and improve of productivity of the domestic animal from transgenic animal is the major technology of biotechnology. Lactoferrin has its highest content in the colostrum, and is known as the antiviral substance. Insulin like growth factor1 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 insulin like growth factor1 gene is inserted into the β-casein gene locus for expression of bovine insulin like growth factor1 (bIGF1) on the bovine β-casein gene. The knock-in vector consists of 5’ arm region (1.02 kb), insulin like growth factor 1 cDNA, CMV-EGFP, and 3’ arm region (1.83 kb). To express bIGF1 gene as transgene, the F2A sequence was fused to the 5’ terminal of bIGF1 gene and inserted into exon 7 of the β-casein gene. As a result, the knock-in vector is confirmed that the amino acids are synthesized without termination from the β-casein exon 7 region to the bIGF1 gene. These knock-in vectors may help to create transgenic dairy cattle expressing bovine IGF1 protein in the mammary gland via the expression system of the bovine β-casein gene.

The knock-in efficiency in the fibroblast is very important to produce transgenic domestic animal using nuclear transfer. In this research, we constructed three kinds of different knock-in vectors to study the efficiency of knock-in depending on structure of knock-in vector with different size of homologous arm on the β-casein gene locus in the somatic cells; DT-A_cEndo Knock-in vector, DT-A_tEndo Knock-in vector I, and DT-A_tEndo Knock-in vector II. The knock-in vector consists of 4.8 kb or 1.06 kb of 5’ arm region and 1.8 kb or 0.64 kb of 3’ arm region, and neomycin resistance gene(neor) as a positive selection marker gene. The cEndo Knock-in vector had 4.8 kb and 1.8 kb homologous arm. The tEndo Knock-in vector I had 1.06 kb and 0.64 kb homologous arm and tEndo Knock-in vector II had 1.06 kb and 1.8 kb homologous arm. To express endostatin gene as transgene, the F2A sequence was fused to the 5’ terminal of endostatin gene and inserted into exon 7 of the β-casein gene. The knock-in vector and TALEN were introduced into the bovine fibroblast by electroporation. The knock-in efficiencies of cEndo, tEndo I, and tEndo II vector were 4.6%, 2.2% and 4.8%, respectively. These results indicated that size of 3’ arm in the knock-in vector is important for TALEN-mediated homologous recombination in the fibroblast. In conclusion, our knock-in system may help to create transgenic dairy cattle expressing human endostatin protein via the endogenous expression system of the bovine β-casein gene in the mammary gland.