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.

A lacZ reporter gene expression atlas for 313 adult KOMP mutant mouse lines.

Expression of the bacterial beta-galactosidase reporter gene (lacZ) in the vector used for the Knockout Mouse Project (KOMP) is driven by the endogenous promoter of the target gene. In tissues from KOMP mice, histochemical staining for LacZ enzyme activity can be used to determine gene expression patterns. With this technique, we have produced a comprehensive resource of gene expression using both whole mount (WM) and frozen section (FS) LacZ staining in 313 unique KOMP mutant mouse lines. Of these, ∼ 80% of mutants showed specific staining in one or more tissues, while ∼ 20% showed no specific staining, ∼ 13% had staining in only one tissue, and ∼ 25% had staining in >6 tissues. The highest frequency of specific staining occurred in the brain (∼ 50%), male gonads (42%), and kidney (39%). The WM method was useful for rapidly identifying whole organ and some substructure staining, while the FS method often revealed substructure and cellular staining specificity. Both staining methods had >90% repeatability in biological replicates. Nonspecific LacZ staining occurs in some tissues due to the presence of bacteria or endogenous enzyme activity. However, this can be effectively distinguished from reporter gene activity by the combination of the WM and FS methods. After careful annotation, LacZ staining patterns in a high percentage of mutants revealed a unique structure-function not previously reported for many of these genes. The validation of methods for LacZ staining, annotation, and expression analysis reported here provides unique insights into the function of genes for which little is currently known.

Reporter Gene Silencing in Targeted Mouse Mutants Is Associated with Promoter CpG Island Methylation.

Targeted mutations in mouse disrupt local chromatin structure and may lead to unanticipated local effects. We evaluated targeted gene promoter silencing in a group of six mutants carrying the tm1a Knockout Mouse Project allele containing both a LacZ reporter gene driven by the native promoter and a neo selection cassette. Messenger RNA levels of the reporter gene and targeted gene were assessed by qRT-PCR, and methylation of the promoter CpG islands and LacZ coding sequence were evaluated by sequencing of bisulfite-treated DNA. Mutants were stratified by LacZ staining into presumed Silenced and Expressed reporter genes. Silenced mutants had reduced relative quantities LacZ mRNA and greater CpG Island methylation compared with the Expressed mutant group. Within the silenced group, LacZ coding sequence methylation was significantly and positively correlated with CpG Island methylation, while promoter CpG methylation was only weakly correlated with LacZ gene mRNA. The results support the conclusion that there is promoter silencing in a subset of mutants carrying the tm1a allele. The features of targeted genes which promote local silencing when targeted remain unknown.

Fluorescence-based multiplex real-time RT-PCR arrays for the detection and serotype determination of foot-and-mouth disease virus.

This study reports the development of distinct fluorescence-based multiplex rRT-PCR arrays, which are comprised of unique primer pairs that are statistically modeled from publicly available sequences, for overall foot-and-mouth disease virus and serotype-specific detection. Performance of the multiplex pan FMDV (MpFMDV) array was compared to the 5' UTR and two 3D(pol) assays, following tests on the Miniopticon (BioRad) and 7900HT SDS (ABI) platforms. The MpFMDV array and a recently developed alternative 3D(pol) assay correctly identified all 31 isolates that represented all seven serotypes, including 10 isolates reported previously to be undetectable by both OIE recommended assays (5' UTR and 3D(pol) assay). Statistically significant differences in mean C(T) values were observed with both platforms. With the Miniopticon, the MpFMDV assay detected FMDV at 9.86 and 2.67 cycles less than the 5' UTR and one of the 3D(pol) assay, respectively; whereas with the 7900HT SDS, the MpFMDV assay detected FMDV at 7.11, 4.71, and 2.33 cycles less than the 5' UTR and both 3D(pol) assays, respectively. The MpFMDV assay was more sensitive than the 5' UTR assay, which had a higher mean endpoint dilution of 1.3 log(10). With the exception of the serotype A and C multiplex arrays, the multiplex serotype-specific arrays correctly identified all isolates from five of seven serotypes (O, Asia 1, SAT 1, 2, and 3). Results presented in this study provide proof-of-principle and bench validation for the primer design model, and for the ability of multiplex arrays to accurately detect FMDV and to provide some degree of serotype discrimination of FMDV.