Pancreatic Organoid Lentiviral Transduction

Pancreatic organoids infection was performed as described in^{21 (link)}. Viruses were produced in HEK293T cells and used at multiplicity of infection of 10 for the transgene or 20 for the transctivator Tet-On® 3G. Organoids were dissociated into small clusters, incubated with culture media containing the viruses, 8 μg/ml polybrene (Sigma), 10 μM ROCKi (Sigma) and span for 1 h at 300 × G at room temperature. After the spin, infected organoids were incubated in a cell culture incubator at 37 °C for 5–6 hours before plating in matrigel with fresh media supplemented with ROCKi.

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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

polybrene ROCKi

Cell culture Cells Culture media Infection Matrigel Organoids Pancreatic Polybrene Transgene Viruses

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

Multiplicity of infection (MOI) of the transgene (10)
Multiplicity of infection (MOI) of the transactivator Tet-On® 3G (20)
Incubation time with the viruses (5-6 hours)

dependent variables

Infection of pancreatic organoids

control variables

Polybrene concentration (8 μg/ml)
ROCKi concentration (10 μM)
Centrifugation speed (300 × G)
Centrifugation duration (1 hour)
Centrifugation temperature (room temperature)

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

Annotations

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