Application of inter-simple sequence repeat PCR to mouse models: assessment of genetic alterations in carcinogenesis.
Genomic instability is believed to play a significant role in cancer development by facilitating tumor progression and tumor heterogeneity. Inter-simple sequence repeat (inter-SSR) PCR has been proved to be a fast and reproducible technique for quantitation of genomic instability (amplifications, deletions, translocations, and insertions) in human sporadic tumors. However, the use of inter-SSR PCR in animal models of cancer has never been described. This new technique has been adapted in our laboratory for the analysis of spontaneous and induced mouse tumors. We established the best PCR conditions for each microsatellite-anchored primer and critically evaluated the reproducibility of the band patterns. We also studied the variation of the fingerprints between and within various inbred mouse strains, including wild-derived lines. Tumor-specific alterations were detected as gains, losses, or intensity changes in bands when compared with matched normal DNA. We quantitated the extent of alterations by dividing the number of altered bands in the tumor by the total number of bands in normal DNA (instability index). By means of inter-SSR PCR, we successfully analyzed genomic alterations in various mouse tumors, including spontaneous thymic lymphomas developed in Msh2 knockout mice as well as chemically induced squamous cell carcinomas and thymic lymphomas. Instability index values ranged between 0 and 9%, the highest levels observed in N-methyl-N-nitrosourea-induced thymic lymphomas generated in Trp53 (p53) nullizygote (-/-) mice. We report here, for the first time, the use of inter-SSR PCR to detect somatic mutations in mouse tumoral DNA, including laser-capture microdissected, methanol-fixed tissues. These PCR-based fingerprints provide a novel approach to assessing the number and onset of mutational events in mouse tumors and will help to understand better the mechanisms of carcinogenesis in mouse models.
Benavides
Fernando
F
Department of Carcinogenesis, The University of Texas M.D. Anderson Cancer Center, Science Park-Research Division, Smithville, Texas 78957, USA. fbenavides@sprd1.mdacc.tmc.edu
Zamisch
Mónica
M
Flores
Mónica
M
Campbell
Marcia R
MR
Andrew
Susan E
SE
Angel
Joe M
JM
Licchesi
Julien
J
Sternik
Gabriel
G
Richie
Ellen R
ER
Conti
Claudio J
CJ
D000818:Animals; D003433:Crosses, Genetic; D016172:DNA Fingerprinting / Q000379:methods; D004273:DNA, Neoplasm / Q000032:analysis; D004195:Disease Models, Animal; D051379:Mice; D008806:Mice, Inbred AKR; D008807:Mice, Inbred BALB C; D008809:Mice, Inbred C3H; D008810:Mice, Inbred C57BL; D008811:Mice, Inbred DBA; D016688:Mice, Inbred NOD; D008814:Mice, Inbred NZB; D018632:Mice, Inbred SENCAR; D008815:Mice, Inbred Strains; D008817:Mice, Mutant Strains; D008819:Mice, Nude; D009154:Mutation / Q000235:genetics*; D009368:Neoplasm Transplantation / Q000379:methods; D009369:Neoplasms / Q000139:chemically induced / Q000209:etiology / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics*; D014407:Tumor Cells, Cultured
D004273:DNA, Neoplasm