Generation of gene knockouts and mutant models in the laboratory rat by ENU-driven target-selected mutagenesis.
OBJECTIVE
The rat is one of the most important model organisms for biomedical and pharmacological research. However, the generation of novel models for studying specific aspects of human diseases largely depends on selection for specific traits using existing rat strains, thereby solely depending on naturally occurring variation. This study aims to provide the tools to manipulate the rat genome in a more directed way.


METHODS
We developed robust, automated, and scaleable reverse genetic methodology based on ENU (N-ethyl-N-nitrosourea)-driven target-selected mutagenesis. Optimal mutagenesis conditions have been determined in three different rat strains and a universal, rapid, and cost-effective dideoxy resequencing-based screening setup was established for mutation discovery. The effectiveness of the approach is illustrated by the identification of 120 induced mutations in a set of genes of interest, including six that result in unique rat knockout models due to the introduction of premature stop codons. In addition, 56 mutations were found that change amino acids, including critical residues in transmembrane domains of receptors and channels.


CONCLUSIONS
The approach described here allows for the systematic generation of knockout and protein function altering alleles in the rat. The resulting induced rat models will be powerful tools for studying many aspects of a wide variety of human diseases.
Smits
Bart M G
BM
Hubrecht Laboratory, Utrecht, The Netherlands.
Mudde
Josine B
JB
van de Belt
Jose
J
Verheul
Mark
M
Olivier
Jocelien
J
Homberg
Judith
J
Guryev
Victor
V
Cools
Alexander R
AR
Ellenbroek
Bart A
BA
Plasterk
Ronald H A
RH
Cuppen
Edwin
E
D000477:Alkylating Agents; D000818:Animals; D030801:Animals, Genetically Modified; D004252:DNA Mutational Analysis; D004195:Disease Models, Animal; D005038:Ethylnitrosourea; D005260:Female; D005821:Genetic Techniques; D016678:Genome; D008297:Male; D016297:Mutagenesis, Site-Directed; D010597:Pharmacogenetics / Q000379:methods*; D051381:Rats; D011916:Rats, Inbred F344
D000477:Alkylating Agents; D005038:Ethylnitrosourea