최소 단어 이상 선택하여야 합니다.
최대 10 단어까지만 선택 가능합니다.
다음과 같은 기능을 한번의 로그인으로 사용 할 수 있습니다.
NTIS 바로가기다음과 같은 기능을 한번의 로그인으로 사용 할 수 있습니다.
DataON 바로가기다음과 같은 기능을 한번의 로그인으로 사용 할 수 있습니다.
Edison 바로가기다음과 같은 기능을 한번의 로그인으로 사용 할 수 있습니다.
Kafe 바로가기주관연구기관 | 건국대학교 KonKuk University |
---|---|
보고서유형 | 최종보고서 |
발행국가 | 대한민국 |
언어 | 한국어 |
발행년월 | 2016-01 |
과제시작연도 | 2014 |
주관부처 | 농촌진흥청 Rural Development Administration(RDA) |
등록번호 | TRKO201600003059 |
과제고유번호 | 1395039549 |
사업명 | 국책기술개발 |
DB 구축일자 | 2016-06-25 |
DOI | https://doi.org/10.23000/TRKO201600003059 |
Ⅳ. 연구개발결과
□ 제 1 세부과제 : 초급성/급성/Sall I/적중 형질전환 혹은 HNF-6 형질전환 생쥐 생산 및 기능분석
○ 초급성/급성 KO 모델 생쥐의 기능 분석
- Gal-1, 3 epitope knock-out 생쥐 생산 및 기능 분석
- CMAH knock-out 생쥐 생산 및 기능 분석
○ Multiple KO 생쥐 생산 및 분석
- Gal/CMAH double knock-out 생쥐 생산 및 기능 분석
□ 제 1 협동과제 : 초급성/급성/Sall I 적중 형질전환용 벡터 제작
Ⅳ. 연구개발결과
□ 제 1 세부과제 : 초급성/급성/Sall I/적중 형질전환 혹은 HNF-6 형질전환 생쥐 생산 및 기능분석
○ 초급성/급성 KO 모델 생쥐의 기능 분석
- Gal-1, 3 epitope knock-out 생쥐 생산 및 기능 분석
- CMAH knock-out 생쥐 생산 및 기능 분석
○ Multiple KO 생쥐 생산 및 분석
- Gal/CMAH double knock-out 생쥐 생산 및 기능 분석
□ 제 1 협동과제 : 초급성/급성/Sall I 적중 형질전환용 벡터 제작 및 세포주 개발
○ TALENs를 이용한 세포주 확립
○ CRISPR/Cas9를 이용하여 미니돼지 세포주 확립
□ 제 2 협동과제 : knock-out 세포주의 수립 및 분석
○ CMAH knock-out 돼지 벡터 구축 및 세포주 개발
○ Sal-like 1 knock-out 돼지 벡터 구축 및 세포주 개발
○ pHNF6 knock-out 벡터 돼지 구축 및 세포주 개발
○ Sal-like 1과 pHNF6을 target으로 하는 TALEN 확보
□ 제 3 협동과제 : 초급성/급성/SalI 적중 형질전환 돼지 생산 및 기능 분석
○ CMAH 유전자 적중을 위한 ZFN 벡터 구축 및 형질전환돼지 생산
○ Gal-1, 3/CMAH double knock-out 형질전환돼지 생산
○ Homo화된 Gal-1, 3/CMAH knock-out 형질전환돼지의 기능분석
□ 제 4 협동과제 : 초급성/급성 및 HNF-6 형질전환 돼지 생산 및 무균화
○ 최적의 iPSC 유래 형질전환 세포주 개발 시스템 확립
- 형질전환 세포주를 이용한 iPSC 생산 시스템 구축
- 바이오장기용 돼지의 체세포를 이용한 iPSC 생산 시스템 구축
- Chimera 생산을 위한 배양체계 확립
○ 일반 및 형질전환 세포주를 이용하여 유도만능줄기세포 생산
- 사람의 줄기세포인자를 주입하여 Naive 상태에 가까운 돼지 유도만능줄기세포 생산
○ GalT-KO 형질전환 세포주를 이용하여 유도만능줄기세포 생산
- GalT-KO 돼지 유도만능줄기세포를 생산 및 체외 분화능 검증됨
- 생산된 유도만능줄기세포는 alpha-Gal 발현되지 않음
Recent advancements in gene editing techniques are increasing their availability and application. These techniques, specifically those that incorporate meganucleases, are an attractive alternative to conventional gene targeting methods via homologous recombination due to the ease of use and the high
Recent advancements in gene editing techniques are increasing their availability and application. These techniques, specifically those that incorporate meganucleases, are an attractive alternative to conventional gene targeting methods via homologous recombination due to the ease of use and the high efficiency of gene editing. In this project, first, we successfully produced zinc finger nuclease (ZFN)-mediated monoallelic/biallelic male and female CMP-N-acetylneuraminic acid hydroxylase (CMAH) KO miniature pigs: the efficiency of the gene targeting (41.7%) was higher when donor DNA was used with the ZFN than those of ZFN alone (9.1%). Monoallelic KO pigs had no integration of exogenous DNA into their genome, indicating that this technique would provide a new avenue to reduce the risk of antibiotics resistance when organs from genetically modified pigs are transplanted into patients. Second, to make the GGTA1/CMAH double knock-out (DKO) pig, TALENs designed for the GGTA1 gene were introduced into female CMAH null fibroblast cells with donor DNA, GGTA1 null cells (three cell colonies) were detected. Two colonies (26 & 30) that harbored the same homozygous deletions of 395 bp and the colony(22) that harbored a homozygous deletion of 719 bp of GGTA1 were used to produce the GGAT1/CMAH DKO pigs. A single SCNT was performed, 183 reconstructed embryos were transferred into a single surrogate, and produced 3 piglets. The approach of using donor DNA in conjunction with a meganuclease can be used to increase the efficiency of gene targeting. The gene editing methods can be applied to other genes as well as other mammalian systems. Additionally, we predict that the utility of the CMAH/GGTA1 DKO pigs can be a valuable source for the study of pig-to-human xenotransplantation.
□ Functional analysis of CMAH knock-out mice - Gene Expression and Pathway Analysis of Effects of the CMAH Deactivation on Mouse Lung, Kidney and Heart
To identify differential gene expression profiles associated with the loss of Neu5Gc expression, we performed microarray analysis using Illumina MouseRef-8 v2 Expression BeadChip, using the main tissues (lung, kidney, and heart) from control mice and CMP-Neu5Ac hydroxylase (Cmah) gene knock-out mice, respectively. Out of a total of 25,697 genes, 204, 162, and 147 genes were found to be significantly modulated in the lung, kidney, and heart tissues of the Cmah null mouse, respectively. In this project, we examined the gene expression profiles, using three commercial pathway analysis software packages: Ingenuity Pathways Analysis, Kyoto Encyclopedia of Genes and analysis, and Pathway Studio. The gene ontology analysis revealed that the top 6 biological processes of these genes included protein metabolism and modification, signal transduction, lipid, fatty acid, and steroid metabolism, nucleoside, nucleotide and nucleic acid metabolism, immunity and defense, and carbohydrate metabolism. Gene interaction network analysis showed a common network that was common to the different tissues of the Cmah null mouse.
- MicroRNA Dysregulation in Liver and Pancreas of CMP-Neu5Ac Hydroxylase Null Mice Disrupts Insulin/PI3K-AKT Signaling
CMP-Neu5Ac hydroxylase (Cmah)-null mice fed with a high-fat diet develop fasting hyperglycemia, glucose intolerance, and pancreatic -cell dysfunction and ultimately develop characteristics of type 2 diabetes. The precise metabolic role of the Cmah gene remains poorly understood. This study was designed to investigate the molecular mechanisms through which microRNAs (miRNAs) regulate type 2 diabetes. Expression profiles of miRNAs in Cmah-null mouse livers were compared to those of control mouse livers. Liver miFinder miRNA PCR arrays ( = 6) showed that eight miRNA genes were differentially expressed between the two groups. Compared with controls, seven miRNAs were upregulated and one miRNA was downregulated in Cmah-null mice. Specifically, miR-155-5p, miR-425-5p, miR-15a-5p, miR-503-5p, miR-16-5p, miR-29a-3p, and miR-29b-3p were significantly upregulated in the liver and pancreas of Cmah-null mice.These target miRNAs are closely associated with dysregulation of insulin/PI3K-AKT signaling, suggesting that the Cmah-null mice could be a useful model for studying diabetes.
- Oxidative stress and ROS metabolism via down‐regulation of sirtuin 3 expression in Cmah‐null mice affect hearing loss
CMP‐Neu5Ac hydroxylase (Cmah) disruption caused several abnormalities and diseases including hearing loss in old age. However, underling molecular mechanisms that give rise to age‐related hearing loss (AHL) in Cmah‐null mouse are still obscure. In this study, Cmah‐null mice showed age‐related decline of hearing associated with loss of sensory hair cells, spiral ganglion neurons, and/or stria vascularis degeneration in the cochlea. To identify differential gene expression profiles and pathway associated with AHL,we performed microarray analysis using Illumina MouseRef‐8 v2 Expression BeadChip and pathway‐focused PCR array in the cochlear tissues of Cmah‐null mouse. Pathway and molecular mechanism analysis using differentially expressed genes provided evidences that altered biological pathway due to oxidative damage by low expressed antioxidants and dysregulated reactive oxygen species (ROS) metabolism. Especially, low sirtuin 3 (Sirt3) gene expressions in Cmah‐null mice decreased both of downstream regulator (Foxo1 and MnSod) and regulatory transcription factor (Hif1α and Foxo3a) gene expression. Taken together, we suggest that down‐regulation of Sirt3 expression leads to oxidative stress and mitochondrial dysfunction by regulation of ROS and that it could alter various signaling pathways in Cmah‐null mice with AHL.
□ Production of monoallelic and biallelic CMAH knock-out miniature pigs
After screening KO events from each colony derived from single cells, multiple cell colonies were used to produce CMAH KO pigs. Male C3 cells harboring a ZFN induced mutation through NHEJ on CMAH gene, male D11 and female D1 cells shown to have a biallelic modification of CMAH, and the other cell lines had a monoallelic modification of CMAH were used to produce CMAH KO pigs. By using male C3 cells, SCNT-produced 13 piglets carrying a 1 bp insertion without insertion of donor DNA on one CMAH allele.Also, 5 miniature pigs carrying mono- or biallelic modification of CMAH gene were produced when ZFN with donor DNA was used for gene targeting. In this project, the average efficiency of SCNT was 0.9% (Table 3) and, the CMAH KO pigs reached sexual puberty and are fertile with no sign of abnormalities.
□ Production of biallelic GGTA1 Knock-out Miniature Pigs in a CMAH-null Background
Upon screening for GGTA1 KO events by both flow cytometry and PCR, three cell colonies were used to produce CMAH/GGTA1 DKO pigs. Two colonies (26 & 30) that harbored the same homozygous deletions of 395 bp and the colony (22) that harbored a homozygous deletion of 719 bp of GGTA1 were used to produce the CMAH/GGAT1 DKO pigs. A single SCNT was performed, 183 reconstructed embryos were transferred into a single surrogate, and produced 3 piglets. Genotyping from the cloned pigs showed the expected deletions of the GGTA1 and CMAH genes. Two piglets harbored a 395 bp deletion, one piglet had a 716 bp deletion, and for all three piglets, the genotypes were biallelic modifications for the respective deletions.
□ Decreased transcript level of H-D and Tn-related Genes in CMAH & GGTA1 DKO Pig Cells
The Hanganutziu-Deicher antigen (H-D) plays a pivotal role in acute immune rejection of pig xenografts (37-39). Generally, H-D antigen families are classified as 2 different subfamilies; ST3Gal (ST3Gal1, ST3Gal2, ST3Gal3, ST3Gal4, ST3Gal5, and ST3Gal6) and ST6Gal (ST6Gal1 and ST6Gal2), according to the carbohydrate linkages synthesized (40). To normalize the mRNA level of WT-, CMAH biallelic KO-, and CMAH/GGTA1 biallelic DKO-pigs, ACTB mRNA was used as an internal standard. After normalization with ACTB mRNA, ST3GAL1, ST3GAL3, ST3GAL4, ST6GAL1, and ST6GAL2 gene expression in CMAH/GGTA1 biallelic DKO-pigs were significantly down-regulated, whereas ST3GAL2 was up-regulated, compared to those of WT and CMAH biallelic KO pigs, respectively. This observation suggested that down-regulated ST3GAL1, ST3GAL3, ST3GAL4, ST6GAL1, and ST6GAL2 in CMAH/GGTA1 biallelic DKO-pigs potentially should result in decreased Siaα2,3Galβ1,3GalNAc-R, Siaα2,3Galβ 1,4GlcNAc-R and Siaα2,6Galβ1,4GlcNAc-R expression, but increased Siaα2,3Galβ 1,3GalNAc-R, Sialyl Lew X: Siaα2,3Galβ1, 4(Fucα1,3)GalNAc-R, and Siaα2,4Galβ 1,4GlcNAc-R expression, respectively, that act as an immune antigen within transplanted recipients. Also, ST6GALNAC3 expression for Sialyl-Tn antigen, GALNT2, GALNT3, GALNT4 and GALNT7 expression for Tn antigen in biallelic CMAH KO pigs were significantly down-regulated compared with CMAH biallelic KO pigs.
과제명(ProjectTitle) : | - |
---|---|
연구책임자(Manager) : | - |
과제기간(DetailSeriesProject) : | - |
총연구비 (DetailSeriesProject) : | - |
키워드(keyword) : | - |
과제수행기간(LeadAgency) : | - |
연구목표(Goal) : | - |
연구내용(Abstract) : | - |
기대효과(Effect) : | - |
Copyright KISTI. All Rights Reserved.
※ AI-Helper는 부적절한 답변을 할 수 있습니다.