{"id":{"repo_id":"ajou","oai_identifier":"oai:repository.ajou.ac.kr:201003/11813"},"canonical_url":"https://search.dev.ndltd.org/etd/ajou/oai:repository.ajou.ac.kr:201003/11813","repository":{"repo_id":"ajou","name":"Ajou University","base_url":"http://repository.ajou.ac.kr/oai/request"},"display":{"title":"Expanding the genetic code of a mouse","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.","abstract_html":"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.","abstract_has_math":false,"creators":["이, 순장"],"institution":null,"degree_name":null,"degree_level":null,"degree_discipline":null,"degree_department":null,"school":null,"contributors":["박, 찬배","대학원 의생명과학과","201324380"],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2015,"date_issued":"2015-10-26","date_published":"2015-10-26","updated_at":"2026-07-24T00:51:50Z","subjects":["aminoacyl-tRNA synthetase","Acetyllysine","pyrrolysyl-tRNA","nonsense-mediated mRNA decay"],"languages":["en"],"rights":[],"rights_urls":[],"identifier_entries":[{"key":"dc:identifier","label":"Identifier","values":["http://dcoll.ajou.ac.kr:9080/dcollection/jsp/common/DcLoOrgPer.jsp?sItemId=000000020382","000000020382"],"render_values":[{"text":"http://dcoll.ajou.ac.kr:9080/dcollection/jsp/common/DcLoOrgPer.jsp?sItemId=000000020382","href":"http://dcoll.ajou.ac.kr:9080/dcollection/jsp/common/DcLoOrgPer.jsp?sItemId=000000020382","code":true},{"text":"000000020382","href":null,"code":true}]}]},"links":{"outbound_url":"http://repository.ajou.ac.kr/handle/201003/11813","outbound_label":"Repository record","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["박, 찬배","대학원 의생명과학과","201324380","이, 순장"]},{"key":"dc:creator","label":"Author","values":["이, 순장"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date","label":"Dc Date","values":["2015-10-26","2015"]},{"key":"dc:type","label":"Dc Type","values":["Thesis","Theses"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["aminoacyl-tRNA synthetase","Acetyllysine","pyrrolysyl-tRNA","nonsense-mediated mRNA decay"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["en"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["http://repository.ajou.ac.kr/handle/201003/11813","http://dcoll.ajou.ac.kr:9080/dcollection/jsp/common/DcLoOrgPer.jsp?sItemId=000000020382","000000020382"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description","label":"Description","values":["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.","I.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","Master"]},{"key":"dc:title","label":"Title","values":["Expanding the genetic code of a mouse","마우스의 확장된 유전자 코드 연구"]}]}],"canonical_facts":{"dc:contributor":["박, 찬배","대학원 의생명과학과","201324380","이, 순장"],"dc:creator":["이, 순장"],"dc:date":["2015-10-26","2015"],"dc:description":["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.","I.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","Master"],"dc:identifier":["http://repository.ajou.ac.kr/handle/201003/11813","http://dcoll.ajou.ac.kr:9080/dcollection/jsp/common/DcLoOrgPer.jsp?sItemId=000000020382","000000020382"],"dc:language":["en"],"dc:subject":["aminoacyl-tRNA synthetase","Acetyllysine","pyrrolysyl-tRNA","nonsense-mediated mRNA decay"],"dc:title":["Expanding the genetic code of a mouse","마우스의 확장된 유전자 코드 연구"],"dc:type":["Thesis","Theses"]},"updated_at":"2026-07-24T00:51:50Z"}