Calcitonin (CT) is a small peptide hormone produced primarily in thyroidal C-cells and to a lesser extent in other cell types. Despite the fact that it is a potent inhibitor of bone resorption, debate persists over a physiologic role for this peptide. It has not been possible to create a model of absolute calcitonin deficiency because of its expression in widely-dispersed cells. The gene encoding calcitonin contains 6 exons and the primary RNA transcript is alternatively processed to produce a second peptide, calcitonin gene- related peptide (CGRP). Processing occurs in a cell-specific manner with production of a mRNA encoding CT in thyroidal C- cells and CGRP in neuronal cells. We propose to inactivate the calcitonin locus by insertion of a neomycin resistance gene into exon 3 or 4 of the CT gene. The selection of an appropriate vector is complicated by alternative processing of the primary RNA transcript. We propose to inactivate the calcitonin locus in embryonic stem cells by insertion of a neomycin resistance gene into exon 3 or 4 of the calcitonin gene utilizing homologous recombination techniques. This targeting strategy is likely to eliminate CT and CGRP production. An alternative strategy will utilize a two-step process to create a frame- shift mutation in exon 4 which will create a stop codon and not interfere with normal RNA processing events. We anticipate CGRP will be produced normally in those cell types which process the pre mRNA to produce CGRP mRNA. Mutations will be studied in in vitro and in vivo RNA processing systems prior to utilization to exclude an unanticipated effect on RNA processing. Embryonic stem cells recombinant for the chimeric gene will be selected by growth in G418- and FIAU-containing medium, cloned and established by standard techniques and a calcitonin- deficient animal created by mating of heterozygous parents. Proposed studies of homozygous animals include analysis of bone function and resorption, renal function, studies of CNS function and sexual function.
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