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

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

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

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

Spatial organization of up- and down-regulated genes flanking DEL and TRAP targets in HOM mutant mouse tissues.

<p>Genomic distances from the target are binned into 50 kb intervals with negative numbers for genes 5-prime of the target and positive numbers for genes 3-prime of the target. A total of 113 dysregulated genes are represented in these plots, not including the targeted gene.</p

Percent of DEL and TRAP targets and libraries with one or more up- or down-regulated genes within 500kb of the target.

<p>A chi square statistic was used to compare the frequency of dysregulated genes between the two allele types. NS = not significant with p > 0.05.</p

Regional effects of gene targeting in kidney of TRAP <i>Fbxo44</i> mutant (Top Panel), in spleen of DEL <i>G6b (AU023871)</i> mutant (Middle Panel), and in gonadal adipose tissue of DEL <i>Tst</i> mutant (Bottom Panel).

<p>The genes listed on the X-axis are all found in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005691#pgen.1005691.s001" target="_blank">S1 Table</a> for each targeted gene. In HOM <i>Fbxo44</i> kidney, flanking gene <i>Fbxo6</i> is significantly down-regulated relative to WT controls. In HOM <i>G6b</i> lung, two flanking genes are down-regulated, while three genes 3’ to <i>G6b</i> are up-regulated. In HOM <i>Tst</i> adipose tissue, gene <i>Tex33 3’</i> to <i>Tst</i> is highly up-regulated, while <i>Mpst1</i> 5’ and <i>Kctd17</i> are down-regulated. Gene symbols are listed in centromere to telomere order from left to right without consideration of distance or spacing. The total interval depicted in each graph is +/- 500 kb flanking the centrum of the targeted gene. Asterisks indicate a significant dysregulation by the DESeq2 statistic. Arrow indicates direction of targeted gene transcription.</p

Reads for <i>Arap1</i> TRAP HOM and WT liver, brown adipose (interscapular), abdominal muscle, gonadal white adipose and lung mapping to <i>Arap1</i> genomic sequence.

<p>Exons 1 &2 are not shown because either very few or no reads mapped to those exons from the sequenced libraries. The TRAP vector targeting <i>Arap1</i> places the gene trap 5’ to exon 13. Liver and brown adipose tissue have no reads mapping 3’ to exon 12. However, for HOM abdominal muscle, gonadal adipose, and lung, after skipping exons 13, 14, 15 and 16, there are <i>Arap1</i> reads in the more distal exons, albeit at a lower count than those found in WT tissues.</p

Reads for <i>Iqub</i> DEL HOM mutant and WT lung and kidney libraries mapping to <i>Iqub</i> genomic sequence.

<p>The <i>Iqub</i> gene is expressed at low levels in WT lung, and is not expressed in WT kidney. The expression in <i>Iqub</i> DEL HOM mutant lung and kidney is significantly increased compared with WT, but only for exons 9–13. This is consistent with targeting deletion of only exons 1–8 in the DEL mutant, and overexpression of the remaining exons in the mutant.</p

Reads for <i>Slc1a3</i> TRAP mutant and wildtype (WT) control interscapular brown adipose tissue and gonadal adipose mapping to <i>Slc1a3</i> genomic sequence.

<p>In both types of adipose tissue, reads from the mutant libraries map to each exon of the Slc1a3 gene. However, the number of reads is reduced relative to WT. This is a TRAP mutant with the gene trap cassette placed 5’ to exon 2. We found no reads for <i>Slc1a3</i> in libraries created from WT or HOM liver, and very low reads in abdominal muscle, therefore the graphs for those tissues are not presented. This visualization of Tophat2 mapping of RNA-seq read counts to the mm10 mouse reference genome sequence used the IGV reader. Exon segments are cut and pasted into position to show alignment of the reads against the genome reference sequence. At the bottom of each panel is a cartoon of the exons and introns with intronic arrows indicating the direction of transcription. Each panel is labeled with genotype and tissue. The read histograms, or pileups, show the numbers of reads at each nucleotide for each library. The Y-axis read count range of each graph for HOM and WT for the same tissue is adjusted to account for differences in total library reads. Therefore, the height of the bars for each HOM and WT tissue pair indicate the relative abundance of reads.</p

Frequency of up- and down-regulated genes in a genome wide analysis, flanking DEL mutations, and flanking TRAP mutations.

<p>Frequency of up- and down-regulated genes in a genome wide analysis, flanking DEL mutations, and flanking TRAP mutations.</p