Transcriptome Analysis of Targeted Mouse Mutations Reveals the Topography of Local Changes in Gene Expression

Area of Science:

• Genetics and Genomics
• Molecular Biology
• Bioinformatics

Background:

• Gene targeting in mouse models is crucial for studying gene function.
• Unintended consequences, such as off-target effects, can complicate phenotype interpretation.
• Systematic analysis of these effects is needed to improve experimental design.

Purpose of the Study:

• To systematically analyze the frequency and characteristics of unintended gene expression changes (off-target effects) in mouse models with targeted mutations.
• To compare off-target effects between two common gene targeting strategies: targeted trap mutations (TRAP) and deletion alleles (DEL).

Main Methods:

• RNA sequencing (RNA-seq) was used to evaluate gene expression in tissues of 44 homozygous mutant mice and control mice.
• Gene expression was analyzed in targeted genes and neighboring genes within a +/- 500 kb region.
• Two allele types, TRAP and DEL, were assessed, both incorporating a LacZ reporter and neomycin resistance cassette.

Main Results:

• Deletion alleles (DEL) showed significantly higher frequencies of up-regulated neighboring genes compared to TRAP alleles.
• Down-regulated genes were found more frequently than expected genome-wide around both DEL and TRAP targets.
• Transcriptome analysis confirmed successful targeting in 97% of DEL alleles but only 47% of TRAP alleles.

Conclusions:

• Gene targeting strategies, particularly DEL, can induce significant local gene expression changes.
• Understanding these local position effects is critical for accurate interpretation of gene mutation phenotypes.
• Off-target effects vary by targeting strategy and can impact experimental outcomes.