Konstruction and testing of a vector with a sequence for a non – stable Cre recombinase

Skender, Barbara (2015) Konstruction and testing of a vector with a sequence for a non – stable Cre recombinase. Diploma thesis, Faculty of Science > Department of Biology.

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Abstract

The method of lineage tracing in embryology enables identification of cell’s progeny during embryonic development. Method often used in genetic lineage tracing is the use of Cre/loxP recombination system which enables changes in the target gene and introduction of marker gene into the cell. Cre recombinase has high activity and its insertion into cells often leads to spontaneous activation. Consequently, cell tracking is hindered under these conditions. Restriction of activity of Cre recombinase would increase the specificity of the lineage tracing method. The aim of this study was the construction of a vector coding for non – stable Cre recombinas and analysis of its recombination ability. Destabilization of Cre recombinase would be achieved by ligation of PEST sequence, responsible for short half-life of proteins to Cre sequence in pCS2+mCherry-T2A-Cre ERT2 vector . Fusion proteins obtained from this vector have the ability to induce recombination in dependence on addition of tamoxifen, under optimal concentration. Different concentrations of mRNA for stable and destabilised recombinase were in parallel microinjected in one-cell zebrafish embryos (Danio rerio) to optimize concentrations under which Cre/loxP system is inducible by adding tamoxifen. Results shown the appearance of tamoxifen induced recombination in embryo injected with 0,5 pg/μL mRNA for a stable Cre recombinase. There was no difference in effectiveness of recombination between stable and a non - stable recombinase Cre. In order to confirm these results it is necessary to conduct additional testing border concentrations of Cre mRNA in which recombination is inducible (5 – 0,5pg/μl).

Item Type: Thesis (Diploma thesis)
Keywords: zebrafish, PEST, Cre/loxP, Tamoxifen, lineage tracing
Supervisor: Matulić, Maja
Date: 2015
Number of Pages: 31
Subjects: NATURAL SCIENCES > Biology
Divisions: Faculty of Science > Department of Biology
Depositing User: Grozdana Sirotic
Date Deposited: 22 Apr 2015 11:24
Last Modified: 22 Apr 2015 11:24
URI: http://digre.pmf.unizg.hr/id/eprint/3861

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