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Mutagenesis of sepiapterin reductase by imprecise excision of the piggyBac transposon in Drosophila melanogaster
piggyBac transposon의 imprecise excision에 의한 초파리 세피압테린 환원효소 돌연변이체 제작

DC Field Value Language
dc.contributor.advisor임정빈-
dc.contributor.author김희종-
dc.date.accessioned2017-07-14T00:45:26Z-
dc.date.available2017-07-14T00:45:26Z-
dc.date.issued2013-02-
dc.identifier.other000000010060-
dc.identifier.urihttps://hdl.handle.net/10371/121356-
dc.description학위논문 (박사)-- 서울대학교 대학원 : 생명과학부, 2013. 2. 임정빈.-
dc.description.abstractMutagenesis by transposon-mediated imprecise excision is the most extensively used technique for mutagenesis in Drosophila. Although P-element is the most widely used transposon in Drosophila to generate deletion mutants, it is limited by the insertion coldspots in the genome where P-elements are rarely found. The piggyBac transposon was developed as an alternative mutagenic vector for mutagenesis of non-P-element targeted genes in Drosophila because the piggyBac transposon can more randomly integrate into the genome. Previous studies suggested that the piggyBac transposon always excises precisely from the insertion site without initiating a deletion or leaving behind an additional footprint. This unique characteristic of the piggyBac transposon facilitates reversible gene-transfer in several studies, such as the generation of induced pluripotent stem (iPS) cells from fibroblasts. However, it also raised a potential limitation of its utility in generating deletion mutants in Drosophila. In this study, I report multiple imprecise excisions of the piggyBac transposon at the sepiapterin reductase (SR) locus in Drosophila. Through imprecise excision of the piggyBac transposon inserted in the 5′-UTR of the SR gene, I generated a hypomorphic mutant allele of the SR gene which showed markedly decreased levels of SR expression. My finding suggests that it is possible to generate deletion mutants by piggyBac transposon-mediated imprecise excision in Drosophila. However, it also suggests a limitation of piggyBac transposon-mediated reversible gene transfer for the generation of induced pluripotent stem (iPS) cells.-
dc.description.tableofcontentsABSTRACT i
CONTENTS iii
LIST OF FIGURES AND TABLES v
ABBREVIATIONS vii
I. Introduction 1
1. Mutagenesis methods in Drosophila 1
1-1. EMS mutagenesis 1
1-2. Mutagenesis by P-element-mediated imprecise excision 4
1-3. Targeted gene replacement mutagenesis 11
1-4. Knockdown using RNAi 14
2. piggyBac transposon 18
3. Sepiapterin reductase and tetrahydrobiopterin 20
4. Purpose of this study 23
II. Materials and Methods 24
1. Drosophila strains 24
2. Southern blot analysis 24
3. Sequence analysis of piggyBac transposon integration loci 25
4. Induction of piggyBac transposon excision in Drosophila 25
5. Analyses of piggyBac transposon excision type at sepiapterin reductase locus 26
6. RT-PCR 26
7. Real-time RT-PCR 27
8. Sepiapterin reductase assay for Drosophila extracts 27
9. Quantification of content of tetrahydrobiopterin 28
10. Site-directed mutagenesis 28
11. Cloning, expression, and purification of SR and SRT153D, Y166V 29
12. in vitro sepiapterin reductase assay for recombinant proteins 29
13. Generation of transgenic flies 30
14. Immunoblot analysis 30
15. Paraquat-sensitivity assay 31
16. Generation of the SREx234 mosaic clones 31
III. Results 32
1. Characterization of BL18753, a fly line with piggyBac transposon insertion at the sepiapterin reductase locus 32
2. Induction of piggyBac transposon excision in Drosophila 37
3. Identification of piggyBac transposon-mediated imprecise excision 37
4. Characterization of the SREx234 mutant 42
5. Activation of Akt pathway in the SREx234 mutant 49
6. Hyposensitivity of the SREx234 mutant to oxidative stress 58
7. Generation of the SREx234 mosaic clone 58
IV. Discussion 65
V. References 73
ABSTRACT IN KOREAN 86
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dc.formatapplication/pdf-
dc.format.extent2154978 bytes-
dc.format.mediumapplication/pdf-
dc.language.isoen-
dc.publisher서울대학교 대학원-
dc.subject피기백 트랜스포존-
dc.subject초파리-
dc.subject돌연변이-
dc.subject세피압테린 환원효소-
dc.titleMutagenesis of sepiapterin reductase by imprecise excision of the piggyBac transposon in Drosophila melanogaster-
dc.title.alternativepiggyBac transposon의 imprecise excision에 의한 초파리 세피압테린 환원효소 돌연변이체 제작-
dc.typeThesis-
dc.description.degreeDoctor-
dc.citation.pages87-
dc.contributor.affiliation자연과학대학 생명과학부-
dc.date.awarded2013-02-
Appears in Collections:
College of Natural Sciences (자연과학대학)Dept. of Biological Sciences (생명과학부)Theses (Ph.D. / Sc.D._생명과학부)
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