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Secondary iPSC reprogramming to study the role of Wt1 gene

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Secondary iPSC reprogramming to study the role of Wt1 gene

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Wilms tumour suppressor gene (WT1) is essential for normal development of a kidney, it was also discovered as a major controller of mesenchymal-epithelial balance in general development of organs. In this project we use Wt1 and its role in the mesenchymal-epithelial balance to study the mechanism of iPSC reprogramming. Primary iPSCs were generated by the expression of four factors; cMyc, Klf4, Oct4 and Sox2 using a Piggy Bac transposition-based doxycycline-inducible reprogramming system in mouse embryonic fibroblast (MEFs) with NanogKiP and Wt1GFP reporter alleles. Mouse models with NanogKiP and Wt1GFP reporter alleles are used to follow the expression of Nanog and Wt1 gene throughout the reprogramming process. CRISPR-Cas9 has been applied to precisely knockout (KO) the Wt1 gene from one of the selected primary iPSC clone (parental). Chromosome count of primary iPSCs was performed together with a test for their pluripotency and differentiation potential before they were used to produce chimeras to derive secondary MEFs. A secondary iPSC reprogramming was performed on both parental and Wt1 null secondary MEFs. Wt1 was found to be indispensable for iPSC reprogramming, Wt1 null MEFs maintained their mesenchymal morphology throughout the reprogramming process, whereas a significant proportion of wild type MEFs successfully reprogrammed into iPSCs with elevated expressions of pluripotency markers under self-renewal culture conditions.

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OrganisatieHZ University of Applied Sciences
OpleidingChemie
AfdelingDomein Technology, Water & Environment
PartnerLeids Universitair Medisch Centrum (LUMC), Leiden
Datum2019-06-27
TypeBachelor
TaalEngels

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