Saturday, December 21, 2024
12/21/2024
PROJECT SUMMARY This proposal will establish a Molecular Phenotypes of Null Alleles in Cells (MorPhiC) Phase 1: Data Production Research and Development Center based entirely at The Jackson Laboratory for Genomic Medicine – the JAX MorPhiC Center. JAX MorPhiC will comprise, under one roof, a team of highly collaborative scientists with complementary skill sets and decades of cumulative experience in mammalian gene knockouts (KO), stem cell and developmental biology, molecular phenotyping, single cell analysis, and advanced metabolomics and lipidomics capabilities. We will KO 250 protein-coding genes over the Phase 1 period of this initiative and will engineer these KOs in human induced pluripotent stem cells (iPSCs) that will subsequently be differentiated into two cell lineages, the extra-embryonic and the neuroectodermal, where cells will then be comprehensively phenotyped. Our rationale for selecting these lineages is that they are two of the most evolutionary divergent between species, and primate-specificity is one of our criteria for gene prioritization. In addition, the extra- embryonic lineage rapidly develops into a cellular functional endpoint responsible for many biological processes, thus permitting interpretation of function of diverse genes, whereas the early neuroectodermal lineage is relevant to neurodevelopmental disorders. In Aim 1, we will prioritize genes for knockout; selection criteria include expression in extra-embryonic or neuroectodermal lineages, primate-specific features, broad classes of functions while enriching for transcription factors, and genes implicated in human disease. In Aim 2, we will generate iPSC KO clones in high-throughput using high-efficiency protocols and workflows established in JAX's Cellular Engineering core. We will engineer KO clones for 250 genes in the well-characterized, stable human iPSC line, KOLF2.1, while considering the effects of sex, genetic background, possible adaptive/compensatory responses, and different KO strategies including incorporation of conditional/reversible and scalable approaches. In Aim 3, will carry out comprehensive phenotyping of derivatives differentiated from KO iPSCs. We have selected a combination of assays to maximally integrate consistency, scalability, and functional informativeness to help achieve the overall objectives of the MorPhiC Consortium. These include imaging, single cell transcriptomics, single nucleus epigenomics, and metabolomics/lipidomics. Our research Aims will be coordinated in administrative Aim 4, which will ensure efficient project management and oversight, internal and external communications, and data dissemination to the MorPhiC Data Resource and Administrative Coordination Center (DRACC). Successful completion of the proposed work will address several main barriers hampering the ultimate goal of the MorPhiC Consortium to functionally characterize all human genes: identifying the most effective KO strategies and optimal phenotyping technologies, as well as scaling efficiencies to expand this program towards future MorPhiC Phases. In the process, we will generate a valuable resource of 250 human iPSC KO lines that will be distributed to the scientific community via existing operational infrastructure at JAX.
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