Retrotransposon Tto1 as a tool for gene targeting in Arabidopsis thaliana

Popović, Branka (2010) Retrotransposon Tto1 as a tool for gene targeting in Arabidopsis thaliana. Diploma thesis, Faculty of Science > Department of Biology.

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Gene targeting was first applied on mice during the 80-ies of the last century. The success of this strategy is based on a relatively high frequency of homologous recombination in mouse somatic cells and an effective system of positive-negative selection. On the other hand, in plants the integration of exogenous DNA occurs predominantly by illegitimate recombination. In this work we studied the possibility of gene targeting in Arabidopsis thaliana using tobacco retrotransposon Tto1. The idea is based on the manipulation of the process of reverse transcription, a key step in the life cycle of retrotransposon, for production of substrate (cDNA) for homologous recombination. By applying Agrobacterium tumefaciens-mediated transformation several transgenic lines of A. thaliana with different variants of retrotransposon Tto1 were created. Using these lines it was explored whether Tto1 was transcripted into the new host, and whether Tto1 can complete the life cycle with an integrated marker gene. In the case of positive outcome, we will approach to replacing gene marker for a gene that we want to specifically inactivate. It was shown that all variants of cloned Tto1 were transcriptionally active and previously described instability of Tto1 binary vectors in Agrobacterium tumefaciens didn't manifest itself in short-term cultivation. Preliminary results based on gene marker expression, have shown that a Tto1 can complete its life cycle. However, so far new copies were not detected in the genome of transformed A.thaliana plants.

Item Type: Thesis (Diploma thesis)
Keywords: Arabidopsis thaliana, gene targeting, Tto1
Supervisor: Besendorfer, Višnja
Co-supervisor: Malenica, Nenad
Date: 2010
Subjects: NATURAL SCIENCES > Biology
Divisions: Faculty of Science > Department of Biology
Depositing User: Silvana Šehić
Date Deposited: 04 Sep 2014 10:19
Last Modified: 18 Sep 2014 08:23

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