A shift in the percentage of cells expressing GFP was seen in MommeD12
mutants. The percentage of cells was significantly lower than it was in wild-type mice, and the mutants were classified as enhancers of variegation (). The level of mean fluorescence in the expressing cells did not differ between mutants and wild-types (). To identify the mutated genes in MommeD12
, we generated a G2 mapping population by back-crossing MommeD12
mice to Line3C
, a C57BL/6J congenic mouse strain homozygous for the GFP transgene. Mapping by traditional microsatellite and SNP analysis identified a 3.3 Mb interval on chromosome 15 for MommeD12
, A and C). Exome deep sequencing and bioinformatic interrogation of the linked interval, identified a T-to-A transversion in the conserved polypyrimidine tract 10 bp upstream of the 3′ splice site of intron 4 of eIF3h
, a gene not previously considered to play a role in gene silencing (). Sequencing of the cDNA revealed that the mutation leads to skipping of exon 5 (), which results in an in-frame deletion of 50 highly conserved amino acids in the MPN (Mpr1-Pad1-N-terminal) domain.
Figure 1 GFP expression profiles and mean fluorescence of expressing cells in MommeD12 and MommeD38. (A) FACS profiles of MommeD12 and MommeD38 mutants. Erythrocytes from three-week-old mice were analyzed by flow cytometry with a GFP-positive gate set to exclude (more ...)
Figure 2 MommeD12 and MommeD38 have mutations in eIF3h. (A) Schematic of the eIF3h protein structure and positions of point mutations. The point mutation in MommeD12 causes skipping of exon 5. The MommeD38 mutation introduces a premature stop codon at amino acid (more ...)
An Illumina Golden Gate SNP genotyping assay was used to identify the linked chromosome for MommeD38
mapped to an overlapping interval of 22 Mb (Figure S1
, B and C). Subsequent exome deep sequencing and bioinformatic analysis of the interval identified a G-to-A transition in exon 7 of eIF3h
. This mutation changes an arginine to a stop codon ().
For both MommeD12 and MommeD38, putative ENU-induced substitution or indel variants within the linked intervals were identified by comparing exome variant calls generated from the two mutant lines with control exomes. The controls had been prepared in parallel using the same exome capture kits and on the same flow cell to minimize sample-to-sample variation. No other mutations were found in the intervals.
Both mutations lead to reduced eIF3h mRNA (), suggesting that MommeD12 and MommeD38 are null alleles. These are the first mutations reported in this gene in the mouse, and we designated these alleles eIF3hMommeD12 and eIF3hMommeD38.
Heterozygotes for the eIF3hMommeD12
mutations were viable and fertile. However, for both eIF3hMommeD12
, heterozygous intercrosses produced no homozygous offspring at weaning, and timed matings revealed empty deciduas at E9.5 in ratios approximating that expected for the homozygous embryos (). Genotyping of the grossly normal embryos at E9.5 revealed that these were either wild-types or heterozygotes. Intercrosses between eIF3hMommeD12
heterozygotes produced no compound heterozygotes at weaning, as expected (). Our results indicate that eIF3h is required for normal embryonic development in the mouse. This is consistent with the finding that the eIF3h homolog in zebrafish is required for early embryonic development (Choudhuri et al. 2010
Figure 3 Embryo dissections and heterozygous intercrosses for MommeD12 and MommeD38. (A) Embryonic dissections of eIF3h mutant mice. Embryonic dissections revealed no viable homozygotes at E9.5. All embryos were produced by natural matings, and detection of a (more ...)