초록
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● 후성유전체계 조절단백질(EF)과 4종 전사인자(TF) 간 융합단백질을 발현하는 벡터 제작(총 47종)
● 바이러스 감염을 통한 iPS 세포 유도 기반 기술 확립.
● 4종 전사인자와 융합 시 이들에 의해 ...
● 후성유전체계 조절단백질(EF)과 4종 전사인자(TF) 간 융합단백질을 발현하는 벡터 제작(총 47종)
● 바이러스 감염을 통한 iPS 세포 유도 기반 기술 확립.
● 4종 전사인자와 융합 시 이들에 의해 후성유전체계 변형단백질들의 기능 및 타깃 유전체 부위가 크게 modulation됨을 확인.
● 후성체계조절 융합단백질(EF-TF) 발현으로 유도된 세포내 에피지놈 변형을 정량화함.
● 히스톤 탈메틸화효소(JHDM3A)-Oct4 간 융합단백질 J2/Oct4 발현 시, Oct4 단독 발현 시보다 stem cell 관련 miRNA의 발현이 크게 증가함을 확인(증가된 miRNA 27종 확인).
● ChIP를 통해 Oct4에 의해 발현이 조절되는 타깃 유전자 프로모터 부위로 1)J2/Oct4 융합단백질이 결합, 2)주위 크로마틴의 H3K9me3 수준 감소, 3)H3K4me3 수준을 증가시킴을 확인.
● 히스톤 메틸화효소 Setdb1이 배아줄기세포 분화 시 발현이 크게 감소함을 확인하였으며, co-IP를 통해 Setdb1-Oct4 단백질 간 물리적 결합이 존재함을 확인.
● Setdb1 넉다운 시 DNA메틸화가 크게 감소함을 확인. 특히 DAPI-dense region인 heterochromatin 부위에서의 punctate signals이 현저히 약화된 것을 발견. Setdb1 활용 DNA메틸화조절 가능성 확인.
● iPS 세포의 안정적 유도를 위해 Doxycycline으로 4종 전사인자의 발현이 유도되는 벡터를 가진 형질전환생쥐 제작 및 Doxycycline 처리를 통해 형질전환태아 섬유아세포로부터 iPS 세포 유도함. 후성체계조절 융합단백질(EF-TF) 도입을 통한 iPS 효율 개선 효과 조사(진행 중).
● 체세포에 streptolysin-O(SLO) 처리, 배아줄기세포 추출물 첨가와 ATP 재생액 처리, CaCl2를 이용한 재봉합을 통해 SLO를 이용한 iPS 유도법 확립.
(출처 : 보고서 요약서 3p)
Abstract
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IV. Results
1. Establishment of a 4-(iPS inducible)-factor(TF) expression system(8 vectors)
● Production of 4-factor express...
IV. Results
1. Establishment of a 4-(iPS inducible)-factor(TF) expression system(8 vectors)
● Production of 4-factor expressing retrovial vectos(pLNCX-OCT4, pLNCX-SOX2, pLNCX-KLF4, pLNCX-c-MYC)
● Production of pLNCX-GFP, pLNCX-GFP-OCT4, and mammalian expression vector of GFP-Oct4 and GFP-Sox2.
● Transfection of vectors of above into various cell lines to check their expression
2. Cloning of epigenetic factor(EF) genes and tagging each 4-factor gene(39 vectors)
● JHDM3A(J2), KAT2B(K2), MLL2(M2) cDNA cloning
● Production of mammalian expression and CMV promoter-driven vectors such as mGFP/J2, mGFP/K2, mGFP/M2 and etc.
● Production of 12 types of GFP tagged EF-TF, triple fusion proteins, expression vectors mG/J2/SOX2, mG/J2/OCT4, mG/J2/KLF4, mG/J2/cMYC; mG/K2/SOX2, mG/K2/OCT4, mG/K2/KLF4, mG/K2/c-MYC; mG/M2/SOX2, mG/M2/OCT4, mG/M2/KLF4, mG/M2/c-MYC, and study of their epigenetic impacts on genomes of mammalian cells.
● Production of 12 types of mammalian or retroviral GFP-EF-TF triple fusion protein expression vectors(rmG/J2/SOX2, rmG/J2/OCT4, rmG/J2/KLF4, rmG/J2/cMYC; rmG/K2/SOX2, rmG/K2/OCT4, rmG/K2/KLF4, rmG/K2/c-MYC; rmG/M2/SOX2, rmG/M2/OCT4, rmG/M2/KLF4, rmG/M2/c-MYC)
● Production of 12 types of retroviral EF-TF fusion protein vectors (rJ2/SOX2, rJ2/OCT4, rJ2/KLF4, rJ2/cMYC; rK2/SOX2, rK2/OCT4, rK2/KLF4, rK2/c-MYC; rM2/SOX2, rM2/OCT4, rM2/KLF4, rM2/c-MYC)
3. Establishment of a gene transfer condition of multiple retroviral vectors transfection into somatic cells(Establishment of an iPS induction system by infecting cells with 4-factor expression vectors)
● Using GP2-293, a highly efficient gene transfer system(80-90% efficiency) and a virus production system were established. Also, the most adequate condition for retrovirus concentration by ultracentrifugation was set. Concentrated retrovirus was analyzed by western blot assay with the VSV/G antibody, and infected cells were evaluated by immunostaining, RT-PCR, genomic PCR for successful infection and gene expression. All the steps(transfection, virus production, concentration, infection) were confirmed by molecular-cellular approaches.
● Using the polycistronic pTRE-CKOS(OCT4-SOX2-KLF4-cMYC) vector, virus production and infection condition was studied.
● With mouse embryonic fibroblasts(MEFs) from an Oct4-GFP transgenic mouse, lentivirus, and the polycistronic vectors, iPS colonies were obtained, and GFP expression coincides with Oct4 expression was confirmed.
4. Study of the cloned vectors above for their targets and impacts on the epigenome
● GFP-J2 and GFP-M2 proteins were diffused in both the cytosol and the nucleus, but 4-fator tagging confined them in the nucleus only. - The tagged 4 factors modulated the targets and functions of epigenetic modifying proteins.
● Establishment of a histone demethylation/methylation quantification method : J2 containing vectors showed the most obvious epigenetic changes. Using those vectors, EF reaction level was quantified. Sox2-J2 induced the highest demethylation level of H3K9me3(40.4±8.3; n=203), but Oct4-J2 showed the lowest(4.3±1.3; n=232). Depending on the EF, 10 folds of demethylation level difference was observed. This result reflects the range of each EF target genes.
● Effects of EF-Oct4 and EF-Sox2 ectorpic expression on cullular miRNA profile were researched. The number of expressed stem cell related miRNAs increased 45-67% in EF-Oct4 and EF-Sox2 expressing cells compared to the GFP control. miRNA array showed that expression level of 27 stem cell related miRNAs increased 1.5-7 folds in Oct4-J2 transfected cells than the Oct4-alone ones.
● Chromatin immunoprecipitation(ChIP) analysis for local and global histone modification study : Changes in histone covalent modifications were observed in cells transfected with J2 containing vectors by immunostaining. ChIP result showed GFP-J2-Oct4 was enriched at Oct4 target promoter regions and structurally opened the promoters by modifying histones, for example, lowring H3K4me3 level and raising H3K9me3 level.
● DNA methylation at Oct4 target regions : MS-PCR was conducted with Oct4 target promoters in EF-TF cells to study DNA methylation level changes, but no differences were observed. It is believed that transient expression of EF-TF is not sufficient to detect its effect.
● For an additional candidate epigenetic modifying protein, Setdb1, histone H3K9 demethylase, was chosen, and its usability was tested. Setdb1 expression was observed in a mouse early embryo, blastocyst outgrowth, and a embryonic stem(ES) cells. In differentiating ES cells, Setdb1 expression declined to the undetectable level as Oct4 level did. Co-IP showed Setdb1 and Oct4 bind to each other. In a Setdb1 knock-down cell, changes in histone H3K9 methylation patterns were not observed but decreased DNA methylation level was very apparent. Especially, punctuated signals in DAPI-dense heterochromatin regions became much weaker. We prospected direct modulation of DNA methylation, the most stubborn epigenetic maker, might be possible with Setdb1.
5. Establishment of an iPS cell line with 4-factor-expressing retrovirus and study of the cloned vectors above for their iPS inducibilities
● Since results of virus-infection-based methods vary depending on the titre, establishment of a standardized virus production system is required. For the stable and steady production of iPS, a transgenic mouse with doxycycline(Dox) inducible 4-factor expression gene was produced, and MEFs from its fetuses were isolated.
● The Dox inducible MEFs were transfected with EF-TF vectors, and the efficiency of iPS colony production was studied. Various methods were used to deliver the vectors such viral infection, electroporation, and Lipofectamine.
6. iPS induction with protein extracts of embryonic stem cells
● Apply SLO(streptolysin-O) to somatic cells, Incubate the cells with ES extracts and ATP regeneration buffer, Reseal the cells with CaCl2.
● With the procedure above, the ES protein extract delivery method was established.
● This method was applied to MEFs, and AP positive ES-like colonies were obtained.
(출처 : Summary 8p)
