Analyzing Efficiency of a Lentiviral shRNA Knockdown System in Human Enteroids Using Western Blot and Flow Cytometry

Authors

Adam P Wilson
Karni S Moshal
Addison P Franca
Sasirekha Ramani
Randle Gallucci
Hala Chaaban
Kathryn Y Burge

Publication Date

6-21-2024

Journal

STAR Protocols

DOI

10.1016/j.xpro.2024.103082

PMID

38781076

PMCID

PMC11145376

PubMedCentral® Posted Date

5-22-2024

PubMedCentral® Full Text Version

Post-print

Published Open-Access

yes

Keywords

Humans, Lentivirus, Flow Cytometry, Gene Knockdown Techniques, RNA, Small Interfering, Blotting, Western, Organoids, Genetic Vectors, cell culture, flow cytometry, gene expression, protein expression and purification, cell differentiation, organoids

Abstract

Enteroids are in vitro models to study gastrointestinal pathologies and test personalized therapeutics; however, the inherent complexity of enteroids often renders standard gene editing approaches ineffective. Here, we introduce a refined lentiviral transfection protocol, ensuring sufficient lentiviral engagement with enteroids while considering spatiotemporal growth variability throughout the extracellular matrix. Additionally, we highlight a selection process for transduced cells, introduce a protocol to accurately measure transduction efficiency, and explore methodologies to gauge effects of gene knockdown on biological processes.

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