Determination of transgenic loci by expression FISH

DNA targeting by homologous recombination in mouse embryonic stem (ES) cell s has become a widely used method for manipulating the mouse genome and for studying the role of specific genes in mammalian development. For certain studies, it is necessary to target two or more DNA sequences residing on a particular chromosome. In these situations, it would be important to distin guish whether two sequential gene targeting events in the ES cells have occ urred in cis or in trans. We report here a new application of fluorescence in situ hybridization to RNA molecules present at sites of transcription th at allows the identification of cis and trans gene targeting events in FS c ells. The method is based on detection of transcripts from commonly used se lectable marker genes inserted during homologous recombination. Transcripts are detected in interphase nuclei, making the preparation of mitotic cells unnecessary and obviating the necessity for the more technically demanding DNA detection of genes. The method is applicable to any chromosomal locus, and compared with other methods (e.g., genetic linkage testing in chimeric mice), it will greatly shorten the time required for distinguishing cis an d trans gene targeting events in ES cells. The method also may be useful fo r detecting changes in ploidy of individual chromosomes and loss of heteroz ygosity of genes in single cells in culture and also in animals, for exampl e, during processes such as tumorigenesis. (C) 2001 Academic Press.