Earticle

Genetically modified pigs have been considered valuable models of human disease and donors for xenotransplantation. Here, we used Zinc finger nucleases (ZFNs) to knock out the Yucatan miniature pig α-1,3-galactosyltransferase (GGTA1) gene, which generates Gal epitopes that trigger hyperacute immune rejection in pig-to-human transplantation. ZFNs were designed to cleave a region of the GGTA1 gene. Biallelic GTKO cell lines were established from single cell colonies of ear fibroblasts derived from Yucatan miniature pigs following transfection by electroporation. Two cell lines were selected as donor cell line for somatic cell nuclear transfer (SCNT) for the generation of GTKO pigs. The reconstructed GTKO embryos were subsequently transferred into two recipient gilts, of which one became pregnant. We obtained four live piglets and one stillborn. Genotyping of all cloned individuals was performed. The Gal expression in the fibroblasts of all piglets was analyzed by fluorescence activated cell sorting (FACS) and western blotting. Sequencing analyses of the target site confirmed the homozygous GGTA1-null mutation in all fetuses and piglets, consistent with the genotype of the donor cells. Furthermore, FACS and western blotting analyses demonstrated that Gal epitopes were completely absent from the fibroblasts of all GTKO piglets.

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.

Human dipeptidylpeptidase-4(hDPP-4)는 세포표면에 존재하는 당단백질 수용체이며, 다 양한 세포에 발현되어 염증, 세포이동, 세포분화 등에 관련이 있다고 보고되고 있다. DPP-4억제제는 글루카곤 유사 펩타이드-1(glucagon-like-peptide-1, GLP-1)의 분해를 억제 하여 췌장에서 분비되는 인슐린 양을 조절할 수 있게 개발된 의약품으로 제2형 당뇨병에 많이 사용되고 있다. 이러한 DPP-4 억제제에 대한 체내 안전성을 확인하기 위한 모델동 물로 hDPP-4 유전자가 발현되는 형질전환돼지를 개발하였다. 형질전환동물 생산방법으 로 사용한 수정란 미세주입법은 외래유전자가 염색체 내로 임의적으로 삽입되기 때문에, 삽입된 유전자 수와 게놈 내 삽입 위치에 따른 발현량 차이 및 유전자 간섭 현상이 나타 날 수 있다는 보고가 있어, 본 연구에서는 생산된 hDPP-4 형질전환 돼지의 게놈 유전자 를 분리하여 NGS와 genome walking 방법으로 염색체에 삽입된 외래 유전자인 hDPP-4 에 대한 삽입위치를 분석하고자 하였다. NGS 염기서열 분석 결과, 외래유전자(hDPP-4)와 matching되어 삽입되어 있을 것으로 예상되는 염색체 좌위로 2군데가 추정되었고, 이를 바탕으로 PCR을 통한 추가 분석을 진행하였다. 동시에 기존의 genome walking 방법으 로 염색체를 DraI과 EcoRV로 제한효소로 절단하고, 절단면에 합성된 linker를 붙이고, linker에 특이적인 프라이머로 PCR을 진행하여 증폭산물에 대한 염기서열을 분석하였다. 분석결과 Ch. 1 영역 intergenic 영역에 hDPP-4가 삽입되어 있는 것을 확인할 수 있었다. 이 결과를 바탕으로 삽입된 유전자 수에 대한 분석과 세포막에 발현되는 hDPP-4 발현양 상에 대한 분석을 진행하고자 한다.

이종장기이식을 연구하는 많은 과학자들은 대부분 면역거부 반응 감소 연구에 초첨이 맞춰져 있다. 따라서, hyperacute rejection, acute humoral xenograft rejection, acute cellular xenograft rejection, chronic xenograft rejection과 같은 수용체(recipient)에서의 거부 반응 연구에 집중되어 있는 것이 현실이다. 본 연구에서는 이종에 이식된 심장 자체 또 한 큰 문제점이 발생할 수 있을 것으로 판단되어 연구를 수행하게 되었다. Cynomolgus monkey에 heterotopic 방법을 이용하여 α1,3-Galactosyltransferase 유전자 기능 제거 (GalT-KO) 돼지의 심장을 이종이식 하였다. 이식 후 약 9일간 생존한 원숭이로부터 심장 절편을 채취하여 next generation sequencing 방법을 통해 이종이식을 수행하지 않은 돼 지의 심장과 mRNA의 발현 차이를 분석하였다. 그 결과 이종이식 후 심장에서 심근경색 및 섬유증과 관련된 것으로 알려진 matricellular proteins (Thrombospondin1, Tenascine C, SPARC, Periostin 2C)의 transcripts가 급격히 증가하는 결과를 확인할 수 있었다. 이 러한 결과는 real-time PCR 방법을 이용하여 비교하였을 때, Thrombospondin 1의 경우, 7.183±0.4563배, Tenascine C는 37.67±3.525배, SPARC는 7.662±1.281배, Periostin 2C 는 2.482±0.5193배까지 mRNA의 양이 급격히 증가하는 현상을 재확인할 수 있었으며, 단백질 또한 조직염색을 통해 증가하는 현상을 관찰할 수 있었다. 실제로 이종이식 심장 의 병리적 분석을 통해 심근경색 증상이 확인되었다. 결과적으로 이종장기 이식의 성공 을 위해서 수용동물에서의 면역거부 반응뿐만 아니라, 공여체인 돼지 심장 또한 여러 질 환에 노출되어지고 있으며, 그에 따른 대처가 반드시 필요할 것으로 판단된다.

Pig organs have gotten the spotlight as precious resources which it acts a ‘bridge’ to connect organ donors with recipients of shortage organ problems. The cell mediated immune rejection must overcome to survive animal’s organ in human immune system for successful xenotransplantation. US11 protein originates a CMV and it is translocated MHC Class I molecules in endoplasmic reticulums. The known function of CD55(hDAF) is complement regulation protein which inactivates the c3 cleaving converttase. Also, it regulates released c3 by NK cell. In this study, we have analyzed the significance of US11 and CD55 expression for cell-mediated cytotoxicity. The pig ear fibroblasts obtained to establish transgenic pigs. And genotyping and gene expression confirmed by RT-PCR. Degeneration of MHC Class I evaluate each cell by FACS. Cytotoxicity of NK cell and CD8+ CTLs conducts 1:1, 2.5:1, 5:1 / 10:1, 25:1, 50:1 effector:target ratio, respectively. The expression of US11 has known as to attenuate MHC Class I to prevent cytotoxicity by NK cells and CTLs but our results indicate that make no difference expression of MHC Class I between wild type and US11 cell. Nevertheless, US11 gene products suppress to the interference of NK cells and CD8+ CTLs. In comparison between US11/CD55 cells with US11 cells, CD55 reveals to cause the synergistic effect with US11 gene in cytotoxicity of NK cell. The effect of CD55 expression was significantly interrupting cell lysis by NK cells and CTLs, these reactions are supposed to induce humoral immune activity by cytotoxic cells.

For the pig to human organ transplantation, immune rejection is the biggest problem. It is known that T cell is associated with immune rejection and it is necessary to produce severe combined immune deficiency (SCID) pigs to reduce the rejection. Previous studies show that IL2RG and RAG2 mainly function on T cell development which is mainly functions on immune system. Especially, IL2RG is for the T cell and NK cell development, and RAG2 is for maturation of T and B cells. In this study, to produce IL2RG and RAG2 deficient cell lines, we designed specific sgRNAs which target pig IL2RG and RAG2, respectively, using CRISPR/Cas9 system. Each of the GFP tagged targeting vectors were constructed and transfected into pig fibroblasts, respectively or both, to establish single or double knock-out cell lines. After transfection, GFP positive cells were isolated and many single cells were freely seeded and cultured individually. To analysis genomic types of the cells, we amplified and analyzed the targeting regions on DNA. As a result, 3 knock-out cell lines were established for IL2RG and RAG2, respectively. Also, for the double knock-out cell lines, 1 cell line was analyzed by sequencing. By CRISPR/Cas9 system, we established diverse IL2RG and/or RAG2 knock-out cell lines and the cells are planning on producing SCID pigs.