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Expanding the genetic code of a mouse
|dc.description.abstract||Genetic 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.
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
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
V. CONCLUSION 18
|dc.title||Expanding the genetic code of a mouse||-|
|dc.title.alternative||마우스의 확장된 유전자 코드 연구||-|
|dc.subject.keyword||nonsense-mediated mRNA decay||-|
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