Expanding the genetic code of a mouse

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.

Author dc:creator

• 이, 순장

Contributors dc:contributor

• 박, 찬배
• 대학원 의생명과학과
• 201324380

• aminoacyl-tRNA synthetase
• Acetyllysine
• pyrrolysyl-tRNA
• nonsense-mediated mRNA decay

Language dc:language

en

Identifier

http://dcoll.ajou.ac.kr:9080/dcollection/jsp/common/DcLoOrgPer.jsp?sItemId=000000020382
000000020382

OAI identifier oai:identifier

oai:repository.ajou.ac.kr:201003/11813