Polymerase chain reaction detection of retinoblastoma gene deletions in paraffin-embedded mouse lung adenocarcinomas

Abstract

A polymerase chain reaction (PCR) technique has been developed to detect deletions in the mouse retinoblastoma (mRb) gene using microtomed sections from these paraffin-embedded radiation-induced and spontaneous tumors as the DNA source. Six mRb gene exon fragments were amplified in a 40-cycle, 3-temperature PCR protocol. Absence of any of these fragments relative to control PCR products on a Southern blot indicated a deletion of that portion of the mRb gene. Tumors chosen for analysis were lung adenocarcinomas that were judged to be the cause of death. Spontaneous tumors as well as those from irradiated mice (569 cGy of Co y rays or 60 cGy of JANUS neutrons which have been found to have approximately equal biological effectiveness) were analyzed for mRb deletions. Tumors in six neutron-irradiated mice also had no mRb deletions. However, one of six tumors from Y-irradiated mice (17%) and 6 of 18 spontaneous tumors from unirradiated mice (33%) showed a deletion in one or both mRb alleles. All deletions detected were in the 5' region of the mRb gene. Information obtained from such studies of large numbers of preserved mouse tissues has the potential to aid in the identification of mechanisms of radiation-induced carcinogenesis.