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Expanding the genetic code of a mouse

DC Field Value Language
dc.contributor.author이, 순장-
dc.date.accessioned2015-10-26-
dc.date.available2015-10-26-
dc.date.issued2015-
dc.identifier.urihttp://repository.ajou.ac.kr/handle/201003/11813-
dc.description.abstractGenetic code expansion has used the site-specific insertion of unnatural amino acids into proteins(Greiss and Chin, 2011). An aminoacyl-tRNA synthetase and a tRNA are used to specifically insert the unnatural amino acid during mRNA translation, in response to an amber stop codon (UAG) placed at a user-defined site in a gene interest (Davis and Chin, 2012)

In this study, I used Acetyllysine(AcK) as unnatural amino acids, N? -acetyl-lysyl-tRNA synthetase (AcKRS) as AcK-tRNA synthetase, pyrrolysyl-tRNA(PylT) as tRNA from Methanosarcina mazei (Mukai et al., 2008). Amber codon was inserted in GFP that is role of reporter gene. AcKRS aminoacylates PylT , and mRNA encoding the full-length GFP bearing an amber codon that directs amino acid incopration.

I created AcKRS, GFP mouse, and generated immortalized MEF(mouse embryonic fibroblast). Immortalized MEF(AcKRS.GPF) was treated by AcK., but GFP signal was not detected.

I thought that no dectable GFP signal was likely due to the three reasons : First, AcK did not internalize into the system. Second, AcK can be degraded by deacetylase. Third, mRNA was effected by NMD(nonsense-mediated mRNA decay) that can break mRNA containing amber condons. I find the reason that low GFP expression was due to the degradation of mRNA through NMD

To investigate GFP signal in AcKRS.GFP mouse I did cryosection and observed by confocal microsope. Because NMD efficiency is various according to organs, I got GFP expression in stomach and muscle in AcKRS.GFP mouse.
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dc.description.tableofcontentsI.INTRODUCTION 1

II.MATERIALS AND METHOD 3

A. Generation of transgenic mice 3

B. DNA extraction from mouse tail and genotyping PCR 3

C. Isolation of primary mouse embryo fibroblast and SV40 immortalization 3

D. siRNA Transfection 4

E. Cryosection / Confocal microscope 4

III.RESULTS 5

A. Generation of AcKRS.GFP mouse 5

B. Conformation of transgenic mouse 8

C. No dectectable GFP induced by AcK 9

D. Inhibition of Nonsense-mediated mRNA decay through UPF2 Knock down 12

E. Detection of GFP signal in AcKRS.GFP mouse by cryosection 15

IV.DISCUSSION 17

V. CONCLUSION 18

REFERENCES 19

국문요약 21
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dc.language.isoen-
dc.titleExpanding the genetic code of a mouse-
dc.title.alternative마우스의 확장된 유전자 코드 연구-
dc.typeThesis-
dc.identifier.urlhttp://dcoll.ajou.ac.kr:9080/dcollection/jsp/common/DcLoOrgPer.jsp?sItemId=000000020382-
dc.subject.keywordaminoacyl-tRNA synthetase-
dc.subject.keywordAcetyllysine-
dc.subject.keywordpyrrolysyl-tRNA-
dc.subject.keywordnonsense-mediated mRNA decay-
dc.description.degreeMaster-
dc.contributor.department대학원 의생명과학과-
dc.contributor.affiliatedAuthor이, 순장-
dc.date.awarded2015-
dc.type.localTheses-
dc.citation.date2015-
Appears in Collections:
Theses > Graduate School of Biomedical Sciences > Master
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