Fibroblast cell line with inducible cassette exchange system- tool to study gain and loss of gene function

Bosnakovski, Darko (2012) Fibroblast cell line with inducible cassette exchange system- tool to study gain and loss of gene function. In: 5th International Conference “Biomaterials, Tissue Engineering & Medical Devices” BiomMedD’2012.

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Abstract

We generated fibroblast cell line with single copy of targeting Cre/Lox cassette exchange. The cassette is targeted with circular plasmid P2lox/cre caring the gene of interest. Successful lox/cre recombination provides reconstitution of NEO gene within the cassette that allows positive selection with neomycin. The whole cassette is under doxycyline inducible (TRE) promoter. To evaluate the effectiveness of our inducible fibroblast cell line (i3T3) we recombined the exchange locus with luciferase. The generated luciferase inducible cell line (i3T3-Luciferase) was inducible in time and dose dependent manner to doxycyline treatment. Positive luciferase signal was detected as early as 30 min post induction. Levels of luciferase can be titrated with dilutions of doxycyline from 0.5 to 500 ng/ml. For functionally evaluation of the cell line we recombined the inducible locus with MyoD and Myf5, two main myogenic transcription factors. MyoD and My5 initiated myogenic differentiation in fibroblast. Myogenesis was evident by induction of myogenic pathway and formation of terminally differentiated myotubes. Out inducible cell line allowed us to study direct targets of MyoD and Myf5 and to dissect the process of myogenic reprogramming of fibroblast. We engendered fibroblast cell line with doxycyline inducible cassette exchange system. The cassette can be efficiently targeted with any gene of

Item Type: Conference or Workshop Item (Poster)
Subjects: Natural sciences > Biological sciences
Divisions: Faculty of Medical Science
Depositing User: Darko Bosnakovski
Date Deposited: 01 Feb 2013 12:32
Last Modified: 01 Feb 2013 12:32
URI: http://eprints.ugd.edu.mk/id/eprint/5191

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