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OJVRTM
Online Journal of Veterinary Research©
Volume 19(2): 137-147, 2015.
Development
of human dental pulp stem cells expressing reverse tetracycline-controlled trans activator by different transfection methods
N. Askari1,
N. Karam-Zadeh2, MM. Yaghoobi2*, M. Shamsara1
1National Institute of Genetic Engineering and Biotechnology,
Tehran, 2Department of Biotechnology, Institute of Science and High
Technology and Environmental Sciences, Graduate University of Advanced
Technology, Kerman-Iran
ABSTRACT
Askari N, Karam-ZadehN, Yaghoobi MM, Shamsara M.,
Development of Human Dental Pulp Stem Cells Expressing Reverse
Tetracycline-controlled Trans activator (rtTA) using
Different Transfection Methods, Onl J Vet Res.,, 19 (2):
137-147, 2015. In vivo and in vitro studies suggest that dental pulp stem
cells (DPSCs) display hallmarks of pluripotency and
potential for differentiation toward multiple cells. These properties introduce
them as a new source for cell and gene therapy. Little is known about
transfection efficiency of DPSCs. We report optimization of transfection of
DPSCs by electroporation, calcium phosphate and X-tremeGENE
HP DNA reagents for stable human DPSC line expressing rtTA.
Integration of plasmid and expression of rtTA was
confirmed through antibiotic selection, PCR and RT-PCR. Highest transfection
efficiency by electroporation was achieved at 300 V,2
µg in a 4 pulse square-wave condition. With X-tremeGENE
HP DNA transfection reagent a 3 µl:2µg ratio was optimal whereas with calcium
phosphate an HEPES buffer: DNA ratio of 120 µl:2µg proved ideal. The optimal
time for precipitation of DNA/ calcium phosphate was 2 min. DMSO shock 4 hours
later enhanced transfection efficiency.
Key Words:
Dental Pulp Stem Cells; Tet-On Cell Lines; tet-responsive
transcriptional activator; Transfection.
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