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Transgenic enrichment strategies for cardiac cells derived from mouse embryonic stem cells using HSV1-TK-ganciclovir and CD-5-fluorouracil

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Defense Thèse de doctorat : Univ. Genève, 2013 - Sc. 4542 - 2013/02/05
Abstract The aim of this thesis was to enrich for cardiac cells derived from mouse embryonic stem cells (mESC) during differentiation. In order to achieve this aim we tested two different suicide gene-based systems and used lentivectors as means of transgene delivery into the genome of undifferentiated mESC. One transgenic enrichment approach was based on the use of herpes simplex virus thymidine kinase 1 (HSV1-TK). An enrichment of ~20 fold was obtained and demonstrated using flow cytometry after targeting cardiac troponin T. Enriched cells showed retained cardiac phenotypes as shown via RT-PCR and immunocytochemistry. Spontaneous beating and reactivity to pharmacological stimulation was observed via confocal microscopy after fluo-2 incubation. The second approach based on the suicide gene system composed by cytosine deaminase (CD) and the prodrug 5-fluorocytosine (5-FC). Cardiac enrichment monitored by flow cytometry after staining for cardiac Troponin T reached ~9 fold. Also in this case, enriched cardiac cells retained their cardiac phenotype. In summary, we report the successful enrichment of cardiac cells derived from mESC after lentiviral transduction with either the CD / 5-FC suicide gene system or the HSV1-TK / ganciclovir suicide gene system in combination with the tetR-Krab / tetO2.
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URN: urn:nbn:ch:unige-277304
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HEUKING, Pernilla. Transgenic enrichment strategies for cardiac cells derived from mouse embryonic stem cells using HSV1-TK-ganciclovir and CD-5-fluorouracil. Université de Genève. Thèse, 2013. https://archive-ouverte.unige.ch/unige:27730

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Deposited on : 2013-05-08

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