Abstracts Biological Sciences

Construction of a targeting vector for the analysis of the neuroendocrine function of chromogranin A

by Richard Feldstein

Parathyroid hormone and Chromogranin A (CgA), are the two major secreted products of the parathyroid gland. CgA is the major member of the granin family of acidic glycoproteins which are expressed in all endocrine and neural cells. Granins are thought to play a role in secretory granule formation and targeting of peptide hormones and neurotransmitters to granules of the secretory pathway. The CgA gene is a single copy gene, that has been characterized in human, bovine, mouse and rat where it was found to be composed of eight exons with conserved exon-intron boundaries. It has been found that several of the peptides encoded within the CgA molecule inhibit hormone and neurotransmitter release in either an autocrine or paracrine fashion. The biosynthesis of CgA is regulated by many different factors, including steroid hormones and a number of intracellular messenger systems, such as intracellular calcium, Protein Kinase A (PKA) and Protein Kinase C (PKC). Although a number of studies have been conducted on CgA, the precise role that it plays in neuroendocrine function remains unclear. Therefore, to gain insight into the important functions of CgA, this project involved isolation of the CgA gene through screening of a mouse strain genomic DNA library. Restriction fragments of the phage DNA were subcloned into a plasmid vector, sequenced and were confirmed to contain seven of the eight exons of the mouse CgA gene including, eight kilobases of the 5' flanking region. Through the sequence information obtained, a targeting vector was constructed containing approximately nine kilobases of genomic DNA. This vector in turn, will be used for the functional disruption of the CgA gene in mice through the technique of homologous recombination.