# Analyzing efficiency of a lentiviral shRNA knockdown system in human enteroids using western blot and flow cytometry

## TL;DR

This paper presents an improved method for gene editing in human enteroids using lentiviral shRNA, with protocols for transfection, selection, and efficiency assessment.

## Contribution

A refined lentiviral transfection protocol for human enteroids that accounts for spatiotemporal growth variability and includes methods for measuring transduction efficiency.

## Key findings

- A refined protocol ensures sufficient lentiviral engagement with enteroids.
- A selection process and transduction efficiency measurement method were introduced.
- Methodologies to assess gene knockdown effects on biological processes were explored.

## 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.

•Protocol for lentivirus-based gene editing of gastrointestinal enteroids•Steps for performing a puromycin selection trial•Instructions for assessing transduction efficiency

Protocol for lentivirus-based gene editing of gastrointestinal enteroids

Steps for performing a puromycin selection trial

Instructions for assessing transduction efficiency

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

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.