Targeting of Slc25a21 is associated with orofacial defects and otitis media due to disrupted expression of a neighbouring gene.

Homozygosity for Slc25a21(tm1a(KOMP)Wtsi) results in mice exhibiting orofacial abnormalities, alterations in carpal and rugae structures, hearing impairment and inflammation in the middle ear. In humans it has been hypothesised that the 2-oxoadipate mitochondrial carrier coded by SLC25A21 may be involved in the disease 2-oxoadipate acidaemia. Unexpectedly, no 2-oxoadipate acidaemia-like symptoms were observed in animals homozygous for Slc25a21(tm1a(KOMP)Wtsi) despite confirmation that this allele reduces Slc25a21 expression by 71.3%. To study the complete knockout, an allelic series was generated using the loxP and FRT sites typical of a Knockout Mouse Project allele. After removal of the critical exon and neomycin selection cassette, Slc25a21 knockout mice homozygous for the Slc25a21(tm1b(KOMP)Wtsi) and Slc25a21(tm1d(KOMP)Wtsi) alleles were phenotypically indistinguishable from wild-type. This led us to explore the genomic environment of Slc25a21 and to discover that expression of Pax9, located 3' of the target gene, was reduced in homozygous Slc25a21(tm1a(KOMP)Wtsi) mice. We hypothesize that the presence of the selection cassette is the cause of the down regulation of Pax9 observed. The phenotypes we observed in homozygous Slc25a21(tm1a(KOMP)Wtsi) mice were broadly consistent with a hypomorphic Pax9 allele with the exception of otitis media and hearing impairment which may be a novel consequence of Pax9 down regulation. We explore the ramifications associated with this particular targeted mutation and emphasise the need to interpret phenotypes taking into consideration all potential underlying genetic mechanisms

Disruption of Mouse Cenpj, a Regulator of Centriole Biogenesis, Phenocopies Seckel Syndrome

Disruption of the centromere protein J gene, CENPJ (CPAP, MCPH6, SCKL4), which is a highly conserved and ubiquitiously expressed centrosomal protein, has been associated with primary microcephaly and the microcephalic primordial dwarfism disorder Seckel syndrome. The mechanism by which disruption of CENPJ causes the proportionate, primordial growth failure that is characteristic of Seckel syndrome is unknown. By generating a hypomorphic allele of Cenpj, we have developed a mouse (Cenpjtm/tm) that recapitulates many of the clinical features of Seckel syndrome, including intrauterine dwarfism, microcephaly with memory impairment, ossification defects, and ocular and skeletal abnormalities, thus providing clear confirmation that specific mutations of CENPJ can cause Seckel syndrome. Immunohistochemistry revealed increased levels of DNA damage and apoptosis throughout Cenpjtm/tm embryos and adult mice showed an elevated frequency of micronucleus induction, suggesting that Cenpj-deficiency results in genomic instability. Notably, however, genomic instability was not the result of defective ATR-dependent DNA damage signaling, as is the case for the majority of genes associated with Seckel syndrome. Instead, Cenpjtm/tm embryonic fibroblasts exhibited irregular centriole and centrosome numbers and mono- and multipolar spindles, and many were near-tetraploid with numerical and structural chromosomal abnormalities when compared to passage-matched wild-type cells. Increased cell death due to mitotic failure during embryonic development is likely to contribute to the proportionate dwarfism that is associated with CENPJ-Seckel syndrome

Histopathology reveals correlative and unique phenotypes in a high-throughput mouse phenotyping screen

The Mouse Genetics Project (MGP) at the Wellcome Trust Sanger Institute aims to generate and phenotype over 800 genetically modified mouse lines over the next 5 years to gain a better understanding of mammalian gene function and provide an invaluable resource to the scientific community for follow-up studies. Phenotyping includes the generation of a standardized biobank of paraffin-embedded tissues for each mouse line, but histopathology is not routinely performed. In collaboration with the Pathology Core of the Centre for Modeling Human Disease (CMHD) we report the utility of histopathology in a high-throughput primary phenotyping screen. Histopathology was assessed in an unbiased selection of 50 mouse lines with (n=30) or without (n=20) clinical phenotypes detected by the standard MGP primary phenotyping screen. Our findings revealed that histopathology added correlating morphological data in 19 of 30 lines (63.3%) in which the primary screen detected a phenotype. In addition, seven of the 50 lines (14%) presented significant histopathology findings that were not associated with or predicted by the standard primary screen. Three of these seven lines had no clinical phenotype detected by the standard primary screen. Incidental and strain-associated background lesions were present in all mutant lines with good concordance to wild-type controls. These findings demonstrate the complementary and unique contribution of histopathology to high-throughput primary phenotyping of mutant mice

A gene expression resource generated by genome-wide lacZ profiling in the mouse

Knowledge of the expression profile of a gene is a critical piece of information required to build an understanding of the normal and essential functions of that gene and any role it may play in the development or progression of disease. High-throughput, large-scale efforts are on-going internationally to characterise reporter-tagged knockout mouse lines. As part of that effort, we report an open access adult mouse expression resource, in which the expression profile of 424 genes has been assessed in up to 47 different organs, tissues and sub-structures using a lacZ reporter gene. Many specific and informative expression patterns were noted. Expression was most commonly observed in the testis and brain and was most restricted in white adipose tissue and mammary gland. Over half of the assessed genes presented with an absent or localised expression pattern (categorised as 0-10 positive structures). A link between complexity of expression profile and viability of homozygous null animals was observed; inactivation of genes expressed in ≥21 structures was more likely to result in reduced viability by postnatal day 14 compared with more restricted expression profiles. For validation purposes, this mouse expression resource was compared with Bgee, a federated composite of RNA-based expression data sets. Strong agreement was observed, indicating a high degree of specificity in our data. Furthermore, there were 1207 observations of expression of a particular gene in an anatomical structure where Bgee had no data, indicating a large amount of novelty in our data set. Examples of expression data corroborating and extending genotype-phenotype associations and supporting disease gene candidacy are presented to demonstrate the potential of this powerful resource

Genotype distribution for <i>Slc25a21^{tm1a(KOMP)Wtsi}</i> mice.

<p>From heterozygous inter-crossing, homozygous progeny for <i>Slc25a21^{tm1a(KOMP)Wtsi}</i> were recorded at a sub-Mendelian ratio at post natal day 14 (P14), but a normal ratio at two embryonic time points (E14.5 and E18.5). <i>Slc25a21^{tm1b(KOMP)Wtsi}</i>, <i>Slc25a21^{tm1c(KOMP)Wtsi}</i> and <i>Slc25a21^{tm1d(KOMP)Wtsi}</i> homozygotes were detected at the expected ratio.</p

Auditory brainstem response threshold was elevated in <i>Slc25a21^{tm1a(KOMP)Wtsi}</i> mice.

<p>Auditory brainstem response threshold was elevated across all frequencies in homozygous <i>Slc25a21^{tm1a(KOMP)Wtsi}</i> mice aged 1, 2, 3 and 6 months respectively. Mean ABR thresholds (± SD) are shown for wild-type (green symbols), heterozygote (blue symbols) and homozygote (red symbols) <i>Slc25a21^{tm1a(KOMP)Wtsi}</i> mice (A–D). The click-evoked ABR thresholds (mean ± SD) plotted as a function of age (months) demonstrated a progressive deterioration in <i>Slc25a21^{tm1a(KOMP)Wtsi}</i> mice (E); an observation clearly demonstrated by plotting the mean click-evoked ABR threshold elevation of mutants above wild-types as a function of age (months) (F).</p

Signs of otitis media in <i>Slc25a21^{tm1a(KOMP)Wtsi}</i> mice.

<p>The tympanic membrane viewed from the external ear canal showed an opaque fluid present in <i>Slc25a21^{tm1a(KOMP)Wtsi}</i> homozygous mice (B) that was not present in wild-type controls (A). The opaque exudate was clearly visible on haematoxylin and eosin stained histology sections through the middle ear of <i>Slc25a21^{tm1a(KOMP)Wtsi}</i> homozygous mice (D) compared to wild-types (C). Examination at higher magnification revealed that the exudate containing inflammatory cells including foamy macrophages (solid arrow head), and neutrophils (short arrow) in the middle ear cavity (MEC), and a thickened mucosa (M, double headed arrow) in <i>Slc25a21^{tm1a(KOMP)Wtsi}</i> homozygous mice due to epithelial hyperplasia, oedema and congestion (F). Wild-type mice appeared normal (E).</p

RNA expression of <i>Pax9</i> was reduced in <i>Slc25a21^{tm1a(KOMP)Wtsi}</i> mice.

<p>Quantitative PCR on RNA from E13.5 embryo heads demonstrated that <i>Pax9</i> RNA expression was significantly reduced in <i>Slc25a21^{tm1a(KOMP)Wtsi}</i> (*** p = 1.1E-06) homozygotes, but equivalent to wild-type levels in <i>Slc25a21^{tm1b(KOMP)Wtsi}</i>, <i>Slc25a21^{tm1c(KOMP)Wtsi}</i> and <i>Slc25a21^{tm1d(KOMP)Wtsi}</i> homozygotes.</p

Genotype distribution for <i>Slc25a21^{tm1a(KOMP)Wtsi}</i> mice.

<p>From heterozygous inter-crossing, homozygous progeny for <i>Slc25a21^{tm1a(KOMP)Wtsi}</i> were recorded at a sub-Mendelian ratio at post natal day 14 (P14), but a normal ratio at two embryonic time points (E14.5 and E18.5). <i>Slc25a21^{tm1b(KOMP)Wtsi}</i>, <i>Slc25a21^{tm1c(KOMP)Wtsi}</i> and <i>Slc25a21^{tm1d(KOMP)Wtsi}</i> homozygotes were detected at the expected ratio.</p