Characterization of Retinoblastoma Gene Expression in Canine Tissue and Canine Osteosarcoma

Abstract

The retinoblastoma gene is the prototype for a class of recessive cancer genes in which loss of activity of both normal alleles is associated with the occurrence of cancer. Deletions occurring at the RB1 locus on chromosome 13q14 have been implicated in the formation of retinoblastoma. Deletions of the RB1 locus have also been noted in several cases of osteosarcoma. Mutated genes transcribe a mRNA 4.0kb in length as compared to the normal length of 4.7kb. RNA has been analyzed from canine tissues and OS tumors. Characterizing the mRNA of the RB gene in canine tissues and tumors will allow evaluation of canine osteosarcoma as a molecular model for the human disease and may enable dogs to be used as research models for the benefit of both species. Total RNA was extracted from samples using guanidinium thiocyanate and phenol/chloroform. After quantification, RNA was electrophoresed through horizontal agarose gels. Gels were stained with ethidium bromide and examined for intact RNA. Nucleic acids were transferred overnight to a Nytran membrane. The membranes were hybridized with a 32P-labelled human Rb cDNA and the size of the Rb message analyzed. It was anticipated that a decreased transcript size in some osteosarcoma tissue would indicate that a deletion of the RB gene had occurred. Hybridizations of the Rb probe to RNA from normal tissues revealed a typical transcript of approximately 6kb. Attempts to optimize hybridizations between the Rb cDNA and canine osteosarcoma RNA have been unsuccessful but are continuing.