碩士 / 國立海洋大學 / 水產養殖學系 / 91 / We usually use the microinjection and electroporation method to generate the transgenic fish. Introducing the foreign gene randomly integrated into chromosome by sperm carried transgene and fertilized with the mature eggs or introduced the transgene into the fertilized eggs, cause the transgene mosaic expression or null expression. Using the homologous DNA for recombination and we’ll improve the condition.
In vitro experiment, the transgene could site - specific integration into chromosome in the epithelioma papulosum (EPC) of carp cells, and it also achieved the gene targeting in vivo of mouse .
Rec A protein is a protein of small molecular. It combined with two parts of homologous DNA fragments, and created the heterologous double strain DNA to accelerate the recombination.
There were many problems in the transgenic animals. We could not introduced the transgene and got regular expression of it. Many cases were the “transient expression”. The mosaic transgenic fish even generate null sperm. We hope arise the integration rate of the transgenic animals and achieve the site-specific recombination to solve the problems. It is also valuable of site-specific recombination. For example, the gene interruption to study a chain of cascade functional genes, and we could save the harmed-gene people by gene therapy.
Our goal is site-specific recombination. We used the transgene, pSK+Z6JAKY; pSK+Z9JAKY; pSK+Z18JAKY. It was constructed by zebrafish homologous DNA fragments contain the JAKY (JAK promoter and yellow fin porgy growth hormone gene) cloned in the pSK vector. It is direct introducing into zebrafish fertilized eggs by electroporation. And we were examine the seven group of 21 - 30 days old putative transgenic zebrafish [ No.6 (Rec A binding; non — Rec A binging); No.9 (Rec A binding; non — Rec A binding); No.18 (Rec A binding; non — Rec A binding); JAKY-ZF (non - homologous DNA control, non — Rec A binding)] by nest PCR and transfer DNA to nylon membrane hybridized with DIG-labeled JAKY probe. We find the Rec A protein binded zebrafish homologous transgene (contain JAKY) integration rate reach No.6: 85 %; No.9: 83 %; No.18: 75 %, and non — Rec A binded zebrafish homologous transgene (contain JAKY) integration rate reach No.6: 79.5 %; No.9: 73.7 %; No18: 69 % .The non- homologous DNA (and non — Rec A protein treatment) control constitute only have the 1.8 % integration rate.
| Identifer | oai:union.ndltd.org:TW/091NTOU0086001 |
| Date | January 2002 |
| Creators | Feng, Chi-Ming, 馮繼明 |
| Contributors | Chen, Jau-Der, 陳昭德 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 99 |
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