Tumor clonality is central to any theory of carcinogenesis. Current methods to assess clonality are based on the principle of X-inactivation: random inactivation of genes in either the paternally or maternally derived X-chromosome occurs very early in embryogenesis and it is stably inherited thereafter. Thus, the ability to distinguish between X-linked polymorphic alleles in heterozygote females can be used for tracing. However, since many of the progeny of single stem cells in adult tissues are grouped together forming """"""""patches"""""""", the assumption that a random distribution is attained is not reliable. A large X-inactivation patch size will confound the assessment of tumor clonality, biasing the results towards monoclonality, as recently confirmed in several tissues. An ideal assay should then be independent of cell lineage, able to use both males and females, and easy to perform. We have devised a method to accomplish this in the mouse. Our method is based on the ability to create a random biallelic red or green fluorescent phenotype, in an adult tissue, and therefore completely independent of patch size issues. A cassette encoding a green fluorescent protein (GFP) unit followed by a red fluorescent protein (RFP) in an antisense orientation is surrounded by LoxP sites positioned in opposite orientation. The reporter strain ubiquitously expresses GFP under the strong CMV promoter, giving green fluorescence in every cell type. CRE recombinase is able to """"""""flip"""""""" a piece of DNA when LoxP sites are positioned in opposite orientation. Therefore, when the reporter strain is crossed with a tissue-specific inducible form of CRE, RFP is appropriately positioned for expression, and a switch from green to red fluorescence occurs, both transiently (inducible) and in a specific cell type. Thus, a random biallelic distribution can be generated (either green or red cells) specifically in the tumor cell type to be analyzed. Since this random distribution is generated in the adult, tumor clonality can be assessed without any interference from patch size. Thus, this line can be exploited to reassess clonality in potentially any tumor model, where a tissue-specific promoter is available. In this proposal, we will validate this new approach utilizing a thyroid tumor progression model that we have recently developed in the laboratory. Although clonality assessment is the goal of this proposal, we envision this new reporter mouse strain might become a powerful resource for multiple applications in the future (i.e., line tracing, tumor-stromal/endothelial interactions, monitoring distal metastasis, etc). Therefore, we consider this line will be of interest to other NIH institutes (e.g. NIDDK, NIEHS, NCI) and that will contribute significantly to many areas of research.
