Ana O. Hoff, Philip Catala-Lehnen, Pamela M. Thomas, Matthias Priemel, Johannes M. Rueger, Igor Nasonkin, Allan Bradley, Mark R. Hughes, Nelson Ordonez, Gilbert J. Cote, Michael Amling, Robert F. Gagel
Creation of mice null for the CT/CGRP gene. (a) Schematic representation of CT/CGRP gene tissue-specific alternative RNA processing and precursor peptide processing. (b) Schematic of recombination of targeting vector with CT/CGRP gene. Black bars indicate general location of DNA probes used for Southern screening. (c) Representative Southern analysis of BamHI/XhoI-digested DNA isolated from doubly resistant embryonic stem cells showing targeted (T) and untargeted (U) clones. A restriction map outlining the origin of individual bands is provided in b. (d) Identification of mouse genotype by PCR analysis of tail-derived DNA (see Methods). PCR product in the upper gel shows the presence of normal WT allele (+/+); the lower gel shows rearranged KO allele (–/–). Both alleles are detected in heterozygous animals (+/–). MW, 100-bp ladder molecular weight marker. (e) RT-PCR analysis of mRNA isolated from the thyroid glands of WT or KO animals (see Methods). Primer pairs used detect mRNAs for CT, CGRPα (α) and CGRPβ (β), which have predicted band sizes of 394 bp, 745 bp, and 367 bp, respectively. (f) Calcitonin immunohistochemical staining of thyroid from WT and KO animals (see Methods).