Multicolor Lentiviral Vectors for Overexpression

The coding sequences for mouse Ngn3, Pdx1 and MafA (Addgene: #19412) were cloned into pWPT-eGFP-E2A (for Ngn3), pWPT-LSSmOrange-E2A (for Pdx1) and pWPT-dTomato-E2A (for MafA) and subcloned in the doxycycline inducible lentivirus pLVX_TRE3G vector (Clontech). Ngn3, Pdx1 and MafA were cloned in the multicistronic vector pWPT-eGFP-Poly3TF and subcloned in the lentivirus pLVX_TRE3G vector (Clontech). The doxycycline inducible Tet-On® 3G transactivator (Clontech) was cloned under PGK (phosphoglycerate kinase) promoter. Multicolor and multicistronic lentiviral backbone vectors were kind gift from Dr. Cedric Ghevaert and Dr. Thomas Moreau^{30 (link)}. Phosphomutant Ngn3 was prepared by site directed mutagenesis (QuickChange II Site-Directed Mutagenesis Kit, Stratagene) on serines S14, S38, S160, S174, S183 and S199, as described in^{21 (link)}.

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Azzarelli R., Rulands S., Nestorowa S., Davies J., Campinoti S., Gillotin S., Bonfanti P., Göttgens B., Huch M., Simons B, & Philpott A. (2018). Neurogenin3 phosphorylation controls reprogramming efficiency of pancreatic ductal organoids into endocrine cells. Scientific Reports, 8, 15374.

Publication 2018

pLVX_TRE3G vector

Backbone Coding sequences Doxycycline Lentivirus Mouse Pdx1 Phosphoglycerate kinase Serines Site directed mutagenesis Transactivator Vectors

Corresponding Organization :

Other organizations : University of Cambridge, Hutchison/MRC Research Centre, Max Planck Institute for the Physics of Complex Systems, Wellcome/MRC Cambridge Stem Cell Institute, Medical Research Council, The Francis Crick Institute, Wellcome/Cancer Research UK Gurdon Institute

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Variable analysis

independent variables

Phosphomutant Ngn3

dependent variables

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control variables

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controls

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Negative controls: Not mentioned

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