phenotype of homozygous Aldh1a2gri/gri mutants to be very similar to that of established Aldh1a2 knockout alleles, each exemplifying the abnormalities associated with severe retinoid deficiency (Fig. 1C,F). Mutant Aldh1a2gri/gri embryos die by E10.5 exhibiting a number of striking defects including cessation of growth at ,E8.5–E9.0, defects in axial turning, dilated unlooped hearts, small somites and tiny otic vesicles. Whereas established knock alleles of Aldh1a2 exhibit no axial turning, many Aldh1a2gri/gri mutants display a slightly milder phenotype with a small degree of embryo turning. The slightly milder turning defect may represent a difference of strain background on the axial turning phenotype or may indicate that the Aldh1a2gri allele is not an absolute null. Comparison of the Rdh10trex versus the Aldh1a2gri phenotypes illustrates the less extreme defects elicited by Rdh10trex mutation relative to that of Aldh1a2gri (compare Fig. 1 E, F). At E9.5 Aldh1a2gri/gri mutants have extreme defects in extension and turning of the body axis, with some embryos completely unturned and others with an abnormal half-turned ‘‘J’’ shaped body axis when viewed from the front. In contrast, nearly all E9.5 Rdh10trex/trex embryos are completely turned. Aldh1a2gri/gri mutant embryos lack all pharyngeal arches posterior to the first arch, whereas many Rdh10trex/trex embryos form second arches and, in some cases, rudimentary third arches. The milder phenotype of the Rdh10trex/trex embryos relative to the severe phenotype of Aldh1a22/2 mutants, together with the presence of residual RA activity detected using RARElacZ reporter mice [23], implies the presence of residual RDH function within Rdh10trex/trex embryos. The residual RA in these embryos could result from a small amount of RDH activity remaining for the mutant RDH10 protein, or to the function of unidentified RDH or ADH enzymes. Generation of Rdh10 knockout mice In order to directly evaluate the contribution of RDH10 in embryonic RA synthesis we sought to completely eliminate RDH10 function from developing mouse embryos. To that end, we generated mice for RDH10 activity utilizing ES cells produced by the Chori-Sanger-UC Davis (CSD) arm of the international Knockout Mouse Project (KOMP) consortium. The CSD KOMP knockout construct is designed to create a series of RDH10 Control of RA Synthesis alleles that can be generated by sequential deletion through exposure to Cre or FLP recombinase (Fig. 2A). The initial targeted knock-in construct inserts a splice acceptor-lacZ gene trap cassette between exon 1 and exon 2 of the Rdh10 gene, an allele we call Rdh10bgeo. The gene trap insertion is expected to produce a allele by disrupting the endogenous Rdh10 transcript and producing instead a reporter lacZ fusion transcript. Exposure of the gene trap allele to FLP-recombinase can then be used to excise the disruptive splice acceptor cassette (flanked by FRT sites). FLP- mediated excision of the gene trap cassette creates a functionally wild type/conditional knockout allele of Rdh10 with loxP sites flanking exon 2, an allele we call Rdh10flox. Cre-mediated recombination can then be used to excise the loxP-flanked exon 2, thereby producing a allele we call Rdh10delta. Exon 2 was chosen for deletion because its absence is predicted to create a frameshift mutation thereby truncating any resulting protein product and targeting the transcript for nonsense mediated decay. Prior to distribution, correct targeting of the gene trap construct to the Rdh10 genomic locus in ES cells was assessed by 59 and 39 PCR junction fragment assays at the KOMP production and distribu- tion centers. The quality control tests included long range PCR assays pairing generic targeting cassette primers with Rdh10 locus- specific genomic primers homologous to the sequences upstream and downstream of the target site. In order to generate mice bearing an Rdh10 knockout allele, we obtained from the KOMP consortium ES cell clone Rdh10_D08, which is heterozygous for the Rdh10 gene trap allele. These ES cells were injected into C57BL6/J blastocysts to produce chimeric mice that were identified by PCR amplification of the bgeo fusion gene from tail DNA samples. Male mice chimeric for the Rdh10 gene trap allele were then mated to identify individual animals transmitting the targeted allele. Mice carrying the Rdh10bgeo gene trap allele were crossed with mice carrying ubiquitously expressed FLP recombinase [30] to generate a conditional Rdh10flox allele. Exposure of the gene trap allele to FLP recombinase resulted in excision of the gene trap/bgeo portion of the targeting cassette and leaving a pair of loxP sites flanking exon 2 to create the conditional allele. Rdh10flox mice were then crossed with mice bearing a female germline expressed Cre recombinase [31]. The resulting exposure Figure 2. Targeted Rdh10 gene trap and knockout allele. (A) Schematic diagram of Rdh10 knockout allele obtained as ES cells from KOMP consortium and derivative alleles. For Rdh10bgeo allele, a gene-trap splice acceptor bgeo cassette is introduced between exon 1 and exon 2 of Rdh10 and loxP sites are introduced surrounding exon 2. For the Rdh10flox allele, the gene trap cassette is excised returning function, conditionally, to the Rdh10 gene. For the Rdh10delta allele, exon 2 is deleted by Cre excision of the DNA between the loxP sites. (B) Rdh10 RNA in situ hybridization of E9.5 wild type embryo reveals expression of Rdh10 in specific tissues. Rdh10 RNA staining pattern n.10 embryos. (C) Staining E9.5 Rdh10bgeo embryofor b- galactosidase activity (arrow) reveals gene trap transcript expressed in pattern similar to Rdh10 RNA expression. Rdh10 b-galactosidase staining pattern n.10 embryos. ba, branchial arch; ht, heart; lb, limb bud; op, optic vesicle; psm, presomitic mesoderm; s, somite. doi:10.1371/journal.pone.0030698.g002 PLoS ONE | www.plosone.org 4 February 2012 | Volume 7 | Issue 2 | e30698