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Generierung und Korrektur von Punktmutationen im HPRT-Gen von Säugerzellen mittels modifizierter einzelsträngiger DNA-Oligonukleotide

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vts_5874_7847.pdf (2.201Mb)
96 Seiten
Veröffentlichung
2007-03-26
Authors
Kneisel, Andrea Reinhilde
Dissertation


Faculties
Medizinische Fakultät
Abstract
Targeted correction of a single base in a gene of an eucaryotic cell by specific oligonucleotides is a yet controversial technique. Here, the correction of point mutations is introduced in the hypoxanthine-guanine-phosphoribosyl-transferase (HPRT) gene as an additional model system to test targeted gene correction. In human, Hprt mutations cause Lesch-Nyhan syndrome. Using hamster V79 cells, three cell lines were generated with one hprt point mutation each. These cell lines were treated with specific single-stranded DNA oligonucleotides of 25 and 45 base in length modified with phosphothioate backbones on the four outer bases on each side. The respective hprt point mutation is positioned in the middle of the 45 base single-stranded oligonucleotide which is homologous to the non-transcribed strand of the wildtype hprt. The cells were selected by HAT medium, and the surviving clones were investigated for the correction of the respective hprt mutation. Treatment with the oligonucleotides was successful in repairing all three hprt mutations (hprt cDNA position 74, C-T; position 151, C-T; and position 400, G-A). The correction efficiency was very low but reproducible, with a range from 0,86 to 3,61 successful hprt corrected cells/10^6 cells. It is suggested that this system allows one to investigate targeted gene correction in dependence on the target sequence and the oligonucleotides used.
Date created
2006
Subject headings
[Free subject headings]: Gene repair | Gene targeting | hprt | Point mutation | Single-stranded DNA oligonucleotides | Targeted gene correction | Targeted nucleotide exchange
[DDC subject group]: DDC 610 / Medicine & health
License
Standard (Fassung vom 03.05.2003)
https://oparu.uni-ulm.de/xmlui/license_v1

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DOI & citation

Please use this identifier to cite or link to this item: http://dx.doi.org/10.18725/OPARU-795

Kneisel, Andrea Reinhilde (2007): Generierung und Korrektur von Punktmutationen im HPRT-Gen von Säugerzellen mittels modifizierter einzelsträngiger DNA-Oligonukleotide. Open Access Repositorium der Universität Ulm und Technischen Hochschule Ulm. Dissertation. http://dx.doi.org/10.18725/OPARU-795
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