碩士 / 國立成功大學 / 生物科技研究所碩博士班 / 91 / The technique of delivering a foreign nucleic acid into nuclei is an essential step for research in gene therapy, DNA vaccines and genetically modified organisms. It is usually carried out through physical approaches such as electroporation and microinjection, which brings DNA through both cell and nuclear membranes. However, these physical methods usually have low efficiency and, most importantly, cause damage to the cell or nuclei that leads to undesired side effects.
The DNA delivery method is also important in the development of DNA vaccines. A DNA vaccine was invented around 1990, which can effectively induce immune responses. However, a DNA vaccine must surmount the barriers of both cell membrane and nuclear membranes this is usually carried out through stressful means, such as a gene gun or injection.
We set forth to develop a needle-free DNA delivery system by constructing a fusion protein ( PTD-TOPN ) which consists of a membrane penetration domain that is a tryptophan-rich peptide ( KETWWETWWTEW ), as well as a DNA binding domain that is the N-terminal 200 amino acid residues of the human topoisomerase I. Since the tryptophan-rich peptide has the ability to penetrate a membrane without requiring a specific receptor, and the N-terminal 200 amino acid residues of human topoisomerase I can bind with any DNA regardless the specific nucleotide sequence, we feel that PTD-TOPN has the potential to be a universal DNA delivery vehicle.
We have also demonstrated its feasibility in delivering DNA into nuclei by simply mixing a plasmid that has a GFP gene with the PTD-TOPN and then to COS-7 cells. We found that the GFP gene was expressed in COS-7 cells.
In summary, this fusion protein can be used for any DNA fragment and cells; therefore, it can be a universal method to deliver DNA.
| Identifer | oai:union.ndltd.org:TW/091NCKU5111012 |
| Date | January 2003 |
| Creators | Su-Ching Lin, 林素卿 |
| Contributors | Tzong-Yueh Chen, Huey-Lang Yang, 陳宗嶽, 楊惠郎 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 70 |
Page generated in 0.0188 seconds