Home › Cell Biology › Universal and Efficient Electroporation Protocol for Genetic Engineering of Gastrointestinal Organoids
Cell Biology JoVE (Open Access) Citable · DOI

Universal and Efficient Electroporation Protocol for Genetic Engineering of Gastrointestinal Organoids

DOI: 10.3791/60704-v
What you'll learn
  • ✓ Perform efficient electroporation of gastrointestinal organoids with large plasmids
  • ✓ Assess transfection efficiency across four different organoid types
  • ✓ Complete genetic engineering workflow within a single day using standard buffers
Protocol

Biopharma Insights This protocol describes an efficient electroporation method for the transfection of four different gastrointestinal organoid entities with larger plasmids (to the extent of 10 kB). It can be performed within one day and does not need extensive preparation or special, cost-intensive electroporation buffers.

Difficulty
intermediate
Total time
~1 day per batch
Model organism
Gastrointestinal organoids (intestinal, gastric, colonic)
Biosafety
BSL-1

Steps

1
Prepare gastrointestinal organoids for transfection

Culture and prepare four different gastrointestinal organoid entities from established lines. Dissociate organoids into single-cell suspension suitable for electroporation.

▶ 00:50
2
Electroporate organoid cells with plasmid DNA

Apply electroporation pulse to suspend organoid cells mixed with large plasmids (up to 10 kB) using standard, cost-effective buffers. Complete transfection in a single electroporation step.

▶ 02:23
3
Seed transfected cells into culture wells

Plate electroporated organoid cells into appropriate culture matrices and wells to allow recovery and organoid reformation.

▶ 03:33
4
Quantify transfection efficiency by fluorescence

Measure transfection efficiency using fluorescent marker expression and microscopy to assess genetic modification success across organoid types.

▶ 04:05
5
Compare electroporation results across organoid entities

Analyze and compare transfection efficiency outcomes for the four different gastrointestinal organoid types to validate universal protocol applicability.

▶ 05:18
💬 Comments coming soon