and behavioral defects. The defects remaining in retinal rescued animals indicate that while embryo viability is not dependent on spatially restricted retinal production, proper tissue patterning and development are. With respect to sub-cellular localization of Vitamin A metabolism, the medium chain reductase ADH enzymes are known to function within the cytosol, while the SDR RDHs function in a membrane bound capacity. RDH10 is associated with the membrane-bound compartment [40] and its membrane association is necessary for its function [41]. Recently, we demonstrated that the RDH reaction occurs not in the cytosol where ADHs reside, but in a membrane-bound cellular compart- ment such as that occupied by RDH10 [39]. Within the cytosol, RBP1 binds to retinol and inhibits oxidation. The membrane localization of RDH10 is functionally important, presumably because it allows RDH10 to oxidize RBP1-free pools of retinol available in the membrane compartment (Fig. 6C). These findings suggest that ADHs do not contribute significantly to retinol oxidation because they cannot oxidize the RBP1-bound retinol in the cytosol and are unable to access RBP1-free retinol from membranes (Fig. 6C). They explain why the cytosolic ADHs, although widely expressed, cannot compensate for the loss of RDH10 during embryogenesis. The lack of function of ADHs in embryonic retinol oxidation is consistent with studies of individual and compound Adh knockout mice, which suggest that ADHs function primarily in governing post-natal retinoid homeostasis (reviewed in [35]). In adult mice, ADH1 provides considerable protection against vitamin A toxicity [42,43] whereas ADH4 promotes survival during vitamin A deficiency [44], a difference which illustrates distinct physiological functions for these two enzymes in retinol metabolism. In contrast, ADH3 appears to function redundantly in both the prevention of retinol toxicity and Vitamin A deficiency, a dual role consistent with its ubiquitous expression. Collectively, these biochemical and compound mutant analyses strongly argue against a significant role for ADHs in Vitamin A metabolism and RA synthesis during embryonic development. Given that Rdh10 is the predominant enzyme oxidizing retinol to retinal and the mounting evidence that ADHs make little if any contribution, we conclude that the conversion of retinol to retinal within an embryo must occur predominantly, if not completely, within a membrane-bound compartment. Collectively these data demonstrate the critical roles played by Rdh10 in mediating Vitamin A metabolism and synthesis of RA and as a nodal point in governing the feedback regulation of RA production. Considering the relatively discrete sites of Rdh10 expression, the allelic series of Rdh10 alleles including conditional alleles described here, will facilitate important strategies to characterize the consequences of impaired Vitamin A metabolism and RA synthesis in a precisely controlled spatiotemporal manner. Materials and Methods Generation of Rdh10 conditional mice The Rdh10 mouse strains used for this research project were generated from ES cells obtained from the trans-NIH Knockout Mouse Project (KOMP) Repository, a NCRR-NIH supported strain repository. ES cells were created by CSD consortium from funds provided by the KOMP. To inquire about KOMP products go to www.komp.org, email: or email service@komp.org. The Rdh10_D08 (full clone name, EPD0149_1_D08) ES cell clone was produced by the CSD consortium (Childrens Hospital of Oakland Research Institute, The Sanger Institute, and UC Davis). Quality control for correct targeting was assessed by the KOMP RDH10 Control of RA Synthesis production and distribution centers using PCR based assays that spanned the 59 and 39 junction fragments. The parental ES cell line was JM8.N4, which is a C57BL/6N background. For the mice described in this report, we obtained Rdh10_D08 ES cells from KOMP and injected them into C57BL/6 blastocysts. The Rdh10 gene trap line was thereby established and subsequently maintained on a C57BL6/J background. Some animals, such as those crossed with transgenic reporter lines, were mixed with other backgrounds. The KOMP repository independently generated mice from the same clone of ES cells. The strain name for the KOMP generated mice is C57BL/6N-Rdh10tm1a(KOMP)Wtsi. Aldh1a2gri mutant mice were originally described as grimace mutants obtained as part of a chemical mutagenesis screen for defects in early craniofacial development [29]. Mice carrying the FLP recombinase (FLPeR) in the Rosa26 locus on a C57BL6 background [30] available from the Jackson Laboratory as stock #009086 B6.129S4-Gt(ROSA)26Sortm1(FLP1)Dym/RainJ. Mice car- rying a Cre recombinase transgene expressed in oocytes driven by ZP3 promoter sequences [45] are available from the Jackson Laboratory as stock #003651 C57BL/6-TgN(Zp3-Cre)93Knw. Genotyping All genotyping was accomplished by real-time PCR assays performed by the commercial genotyping service Transnetyx (Transnetyx.com). For the wild type Rdh10 allele assay, a probe denoted ‘‘Rdh10-1 WT’’ was used to detect a fragment spanning the region of the unmodified genomic locus corresponding to the insertion site of the knock in construct. For the Rdh10bgeo allele, an assay was used to detect the presence of lacZ and neomycin, corresponding to the bgeo cassette. For the Rdh10delta allele, a probe denoted ‘‘Rdh10-1EX’’ was used, which detects an amplified region spanning the junction fragment covering the single recombined FRT site and the 39 loxP site. To test for homozygosity of the allele, samples were screened for absence of Rdh10 exon 2 using an assay probe designated ‘‘Rdh10-2 WT’’. The following sequences indicate the relevant amplified regions, excluding primers: Rdh10 wild type allele GCTCATTCATTGCTTGGCTTTGAATGAGTTTAGAT- TGTTGCTTACAGTTTGGATTTGTGAGCCTTAAAGTGAA- TTTC Rdh10delta allele AGTTCCTATTCCGAAGTTCCTATTCTCTAGAAAGTA- TAGGAACTTCGTCGAGATAACTTCGTATAGCATACATT- ATACGAAGTTATGGTCTGAGCTCGCC qPCR To assay for Aldh1a2 expression in Aldh1a2gri mutant embryos, qRT-PCR TaqMan assay was used with the following probe and primers: Aldh1a2Fwd-TGA TAA AAT TCA CGG ATT GAC CAT Aldh1a2Rev-GGG CTC GTG TCT TGT GAA AGT AA Aldh1a2TaqMan Probe - CCTGTAGATGGAGACTAT To assay for Rdh10 expression in wild type and Aldh1a2gri mutant embryos, qRT-PCR SYBR assay was used with the following primers: Rdh10-F: TGG TGT GGT TTC TGG ACA TCA CCT Rdh10-R: CTC TAG CAT CGT TGG AAG AAA GGC To assay for altered Rdh10 mRNA in Rdh10delta mutant embryos, qRT-PCR SYBR assay was used with the following primers: Rdh_q1F: CTGGGACTGTTCAGCACTG Rdh_q1R: TCTACAAGGTAAGGGCAAACC PLoS ONE | www.plosone.org 10 February 2012 | Volume 7 | Issue 2 | e30698