초록 ▼

Ⅳ. 연구개발결과
본 연구 과제를 통하여 생산된 형질전환 복제돼지 생산 효율을 살펴보면 새로 구축한 형질전환 체세포(primary)를 이용하여 생산한 복제난자를 이식한 경우 임신한 8마리의 대리모중에서 5마리가 분만에 성공하였으며 총 12마리의 복제산자를 생산하였다. 또한 생산된 형질전환복제돼지로부터 회수한 태아체세포(fetal cell)를 공여세포로 활용한 경우 5마리가 임신이 확인되었으나 분만까지 진행된 대리모는 1두였으며, 복제산자 1마리가 생산되었다.
생산된 13마리의 형질전환 새끼돼지의 세포를 이용하여 외래 유전

Ⅳ. 연구개발결과
본 연구 과제를 통하여 생산된 형질전환 복제돼지 생산 효율을 살펴보면 새로 구축한 형질전환 체세포(primary)를 이용하여 생산한 복제난자를 이식한 경우 임신한 8마리의 대리모중에서 5마리가 분만에 성공하였으며 총 12마리의 복제산자를 생산하였다. 또한 생산된 형질전환복제돼지로부터 회수한 태아체세포(fetal cell)를 공여세포로 활용한 경우 5마리가 임신이 확인되었으나 분만까지 진행된 대리모는 1두였으며, 복제산자 1마리가 생산되었다.
생산된 13마리의 형질전환 새끼돼지의 세포를 이용하여 외래 유전자 형질전환 여부를 분석한 결과 5마리에서 ɑ-Gal KO 되었음을 확인하였으며, 이들 돼지에서 사람 유래 면역유전 자(DAF, CD39, TFPI, C1 inhibitor 및 TNFAIP3) 적중 여부를 분석한 결과 5마리 모두 적중 되었음을 확인하였다.
생산된 형질전환 복제돼지가 적중된 복합 면역유전자를 정상적으로 번역(translation)하는 지를 확인하기 위하여 단백질 발현을 분석한 결과 2A peptide system으로 인해 5개의 사람 면역관련 유전자가 정상적으로 발현하는 것을 확인하였다.

Abstract ▼

The shortage of human organs as a donor is one of the biggest problems in organ transplantation. One of solutions to resolve the problem is xenotransplantation which is the organ transplantation between other species like pig to human. Even though pig is the most widely used mammal for xenotransplan

The shortage of human organs as a donor is one of the biggest problems in organ transplantation. One of solutions to resolve the problem is xenotransplantation which is the organ transplantation between other species like pig to human. Even though pig is the most widely used mammal for xenotransplantation in that pig is similar with human organ in size and immune system, immune rejection has been arisen for a main problem in pig to human xenograft. Recently, many researchers are trying to overcome the immune rejection with genetically modified pig, but the reason of the rejection is not fully discovered. This study was performed to generate multi-transgenic cloned pigs for xenotransplantation.
To disrupt α-1, 3-galactosyl-transferase (GTKO) which mainly effect to hyperacute rejection, we targeted exon 9 of α-gal using Zinc Finger Nuclease (ZFN) and inserted five immune modulation genes, hCD55, hCD39, hTFPI, hC1 inhibitor, and hTNFAIP3, under the control of endothelial cell lineage-specific promoter, ICAM-2 (2A5-KI). An optimal condition of ICAM-2 promoter was confirmed by luciferase assay and the production of five proteins via 2A-peptide self-processing was confirmed by Western blot. Two GT-2A5-KI cell lines were established in porcine ear skin fibroblast (PEF) cells that were used the donor cells for producing transgenic cloned pigs. In addition, GT-2A5-KI embryos econstructed by SCNT were transferred to surrogate recipients for development to term. Using CRISPR/Cas9 (Cas9), we also constructed two vectors: Cas9-GTKO vector to target exon 4 of α-gal, and 2A4 expression vector that includes hCD55, hCD39, hTFPI, and hC1 inhibitor as modulators of immune-rejection. We co-transfected the two vectors into PEF, and isolated two cell lines using selection markers, neomycin, which is inserted into the two vectors. We verified the establishment of the GTKO-2A4-KI cell lines using Cas9 by PCR and western blot analysis. In this study, 8 surrogates transferred with TG cloned embryos were pregnant and delivered 12 piglets by natural delivery and Caesarian section. And additional 1 re-cloned piglet was born using the TG fetal fibroblast cells originated from the cloned piglet. Among the 13 newborn piglets, the 5 piglets were targeted with ɑ-Gal knock out and knock in human immune genes (DAF, CD39, TFPI, C1 inhibitor, and TNFAIP3).
To measure the translational function of the foreign genes in the 5 piglets, we performed western blotting. The 5 human immune genes were successfully translated and strongly expressed. Unfortunately, the 5 piglets were died within 3 days without specific pathologic symptoms. To investigate the reason of sudden death, complete blood count (CBC) test were performed. According to the test, their blood cells especially immune cells such as WBC, platelet, and neutrophils, etc. were dramatically decreased compared to normal piglets.
In the present study, we don't know exactly which gene is mainly affected the death of the piglets. Although multiple transgenic pigs can be produced using advanced biotechnology techniques, it may not be provided an expected transgenic pig. Despite these results, we verified the establishment of the GTKO-2A4-KI cell lines using Cas9 by PCR and western blot analysis. We expected that transgenic pigs can be developed using the established cell lines by SCNT and the cloned pigs carrying those multiple immune-modulation genes will overcome the current obstacles in xenotransplantation.