Generation of gene knockouts and mutant models in the laboratory rat by ENU-driven target-selected mutagenesis

Bart M G Smits, Josine B Mudde, Jose van de Belt, Mark Verheul, Jocelien Olivier, Judith Homberg, Victor Guryev, Alexander R Cools, Bart A Ellenbroek, Ronald H A Plasterk, Edwin Cuppen

Research output: Contribution to journalArticleAcademicpeer-review

139 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)159-169
Number of pages11
JournalPHARMACOGENETICS AND GENOMICS
Volume16
Issue number3
Publication statusPublished - Mar-2006

Keywords

  • Alkylating Agents
  • Animals
  • Animals, Genetically Modified
  • DNA Mutational Analysis
  • Disease Models, Animal
  • Ethylnitrosourea
  • Female
  • Genetic Techniques
  • Genome
  • Male
  • Mutagenesis, Site-Directed
  • Pharmacogenetics
  • Rats
  • Rats, Inbred F344

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