Phenotyping novel organogenesis lethal KOMP alleles

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Project Summary. Although the mammalian genome has been sequenced for more than 15 years numerous gaps lie in understanding how these sequences function in the context of the organism. The goal of the knock-out mouse project (KOMP) is to functionally annotate the coding sequences of the genome by knocking out individual genes and assessing their role in the mouse. An overarching goal of the project is to determine the role of individual genes in a plethora of complex traits that underlie diseases that pose significant risks to human health including heart disease, diabetes, metabolic syndrome, cognition and high blood pressure. Importantly over 25% of genes knocked out result in embryonic lethality. I currently collaborate on an existing KOMP project designed to examine novel null alleles that are lethal prior to E9.5 and of the 88 examine and only 4 exhibit post-gastrulation defects. The goal of this project is to more effectively use our laboratories expertise in post-gastrulation/early organogenesis to determine the primary defects of 25-40 novel KOMP-derived strains that are critical during these developmental stages. We propose to analyze mutant strains that are present at E9.5 but lethal by E12.5. In Aim 1a, we propose to identify the normal expression pattern of the deleted gene, using either the LacZ reporter allele or in-house generated in situ probes. We will characterize each strain by examining the onset of histological defects in the null embryos/extra-embryonic tissues compared with stage matched controls, and by performing this analysis one day later, when we expect the early phenotype to be more pronounced. In Aim 1b, we will perform a standard 4-color immunofluorescence on null and control embryos, that is designed to detect defects in each of the 3 embryonic germ layers. We anticipate numerous embryonic and extra-embryonic phenotypes and explain how particular phenotypes will be further examined using existing resources in our laboratory. Finally, because we expect that many of the lines examined in Aim 1a and 1b will display primary defects in extra-embryonic tissues, we propose to further investigate the role of 3-5 of these genes in Aim1c by utilizing KOMP-generated conditional ready ES cells to delete that gene in the embryo but retain expression in the extra-embryonic tissues. These conditional experiments, when compared with the null experiments performed in Aim1a and 1b, will allow us to parse out the role of each novel gene in the embryo-proper and in the extra-embryonic tissues. Together these analysis will not only assist with the functional annotation of critical novel genes but will also lead to a better understanding of the etiology of human pediatric diseases and will serve to widen the drug discovery pipeline.