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

David B West,Michael Adkisson,Julia V Kirov,Kent C Lloyd,Pieter J De Jong,Andreanna Cipollone,Eric K Engelhard,Brandon Willis,Jared Rapp,A J Nava

doi:10.1371/journal.pgen.1005691

David B West, Michael Adkisson + Show 8 more

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https://doi.org/10.1371/journal.pgen.1005691

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Abstract

The unintended consequences of gene targeting in mouse models have not been thoroughly studied and a more systematic analysis is needed to understand the frequency and characteristics of off-target effects. Using RNA-seq, we evaluated targeted and neighboring gene expression in tissues from 44 homozygous mutants compared with C57BL/6N control mice. Two allele types were evaluated: 15 targeted trap mutations (TRAP); and 29 deletion alleles (DEL), usually a deletion between the translational start and the 3’ UTR. Both targeting strategies insert a bacterial beta-galactosidase reporter (LacZ) and a neomycin resistance selection cassette. Evaluating transcription of genes in +/- 500 kb of flanking DNA around the targeted gene, we found up-regulated genes more frequently around DEL compared with TRAP alleles, however the frequency of alleles with local down-regulated genes flanking DEL and TRAP targets was similar. Down-regulated genes around both DEL and TRAP targets were found at a higher frequency than expected from a genome-wide survey. However, only around DEL targets were up-regulated genes found with a significantly higher frequency compared with genome-wide sampling. Transcriptome analysis confirms targeting in 97% of DEL alleles, but in only 47% of TRAP alleles probably due to non-functional splice variants, and some splicing around the gene trap. Local effects on gene expression are likely due to a number of factors including compensatory regulation, loss or disruption of intragenic regulatory elements, the exogenous promoter in the neo selection cassette, removal of insulating DNA in the DEL mutants, and local silencing due to disruption of normal chromatin organization or presence of exogenous DNA. An understanding of local position effects is important for understanding and interpreting any phenotype attributed to targeted gene mutations, or to spontaneous indels.

Highlights

In mammalian systems, improvements of transgenic technologies have focused on methods to reduce the influence of surrounding DNA, called position effects at the genomic insertion site, to ensure reliable and consistent expression of the transgene
In this report we evaluate the local effects of TRAP and deletion alleles (DEL) targeting events in C57BL/6N adult mice by using RNA-seq to characterize the expression of the targeted gene, and genes flanking the targeted gene, in tissues from 44 unique mouse lines
When the frequency of up- or down-regulated genes was expressed per library, similar differences were observed with more DEL libraries having both down- and up-regulated genes within 500kb compared with TRAP libraries (Fig 1)

Summary

Introduction

Improvements of transgenic technologies have focused on methods to reduce the influence of surrounding DNA, called position effects at the genomic insertion site, to ensure reliable and consistent expression of the transgene. To this end, strong reliable ubiquitously expressing promoters have been identified and engineered to provide appropriate and consistent transgene expression [1,2,3]. There are a few reports describing unintended position effects of either random integration or targeting events in mammals Most of these reports relate to finding that a random integrant has disrupted either the regulatory domain [e.g., 11–12], or coding sequence of another gene. This lack of data may be a consequence of not looking

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