Vitamin D binding protein (DBP) is the oldest member of the albumin (ALB) and alpha- fetoprotein gene (AFP) multigene family, whose diverse functions include: transport of vitamin D sterols, high affinity binding to actin monomers and participation in the plasma actin scavenger pathway, neutrophil chemotaxis, endotoxin inactivation, fatty acid transport, and macrophage activation. In addition, DBP has been detected on the surface of certain cells, where it may bind via a receptor. The applicants have further postulated that one of its functions may be critical for survival. In past work, the investigators have cloned and characterized DBP cDNAs and genes, and characterized its tissue-specific and developmental patterns of expression.
The Specific Aims of the current project include: characterizing the transcriptional regulation of the DBP gene by functionally mapping deletions of its promoter in CAT assays, identifying nuclear proteins that associate with functionally significant promoter regions by band-shift assays, studying its transcriptional regulation by steroid hormones, and mapping the ALB/AFP/DBP cluster physically and with DNase I.
The second Aim i nvolves testing whether DBP is critical to survival by disrupting the DBP gene via homologous recombination in a mouse. To achieve this Aim, the mDBP gene will be cloned, and fragments of the gene will be combined to generate homologous recombination replacement vectors. These targeting vectors will be transfected into totipotential mouse embryo stem (ES) cells. ES cells selected for homologous recombination will be microinjected into host blastocysts and implanted into foster mothers. By selection of progeny displaying germline chimerism for the disruption of mDBP, followed by judicious mating, a DBP-/DBP- mouse should be obtained. This mouse will be studied to determine if the DBP null phenotype is lethal, and if not, as a model to further characterize DBP's biological functions. A plan to rescue the potential lethal phenotype is further outlined.
The final Aim i nvolves mapping structural domains within the DBP molecule by mutagenesis of DBP cDNA in transcription vectors followed by in vitro transcription/translation. Synthesized proteins will be assayed for their ability to bind to 25(OH)D and actin. These studies will be confirmed in vivo using transgenic technology. These studies are intended to clarify current understanding of DBP gene expression, function, and structure in the context of the most abundantly expressed serum protein gene family.
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