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Updated: Jul 20, 2026

A Protocol for Multiple Gene Knockout in Mouse Small Intestinal Organoids Using a CRISPR-concatemer

A Protocol for Multiple Gene Knockout in Mouse Small Intestinal Organoids Using a CRISPR-concatemer

Published on: July 12, 2017

Generation of multiple knockout mutants using the Cre-loxP system.

Alan R Kimmel¹, Jan Faix

¹Laboratory of Cellular and Developmental Biology, NIDDK, National Institutes of Health, Bethesda, MD, USA.

Methods in Molecular Biology (Clifton, N.J.)

|September 8, 2006

Summary

Generating multiple gene mutations in Dictyostelium discoideum is now easier. A new protocol allows researchers to efficiently create complex mutants by recycling the Blasticidin S marker using Cre recombinase.

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Area of Science:

Cell biology
Developmental biology
Molecular genetics

Background:

Dictyostelium discoideum is a key model organism for cell and developmental biology research.
Genome sequencing and gene disruption techniques have advanced gene function studies.
Overlapping gene functions necessitate the creation of multi-gene mutants.

Purpose of the Study:

To develop a robust system for generating multiple gene mutations in Dictyostelium discoideum.
To provide a protocol for efficient multi-gene disruption using marker recycling.

Main Methods:

Utilized transient expression of Cre recombinase.
Employed recycling of the Blasticidin S selectable marker.
Applied the system to Dictyostelium discoideum.

Main Results:

Successfully generated a system for creating multiple gene mutations.
Demonstrated efficient marker recycling for multi-gene disruption.
Facilitated the study of genes with overlapping functions.

Conclusions:

The developed protocol enables efficient generation of complex Dictyostelium mutants.
This system is valuable for studying gene redundancy and complex biological processes.
Advances research in cell and developmental biology using Dictyostelium.