The development of chromosome-specific composite DNA probes for the mouse and their application to chromosome painting

Abstract

The speed and ease of human cytogenetic analysis has been greatly enhanced by the technique of fluorescence in situ hybridization (FISH). Non-radioactive fluorescently tagged complex DNA probes specific for individual chromosomes can be hybridized to conventionally obtained metaphase chromosome spreads. Several chromosomes may be "painted" concurrently by using combinations of different labeled probes. Surveys of chromosome breakage and rearrangement may be performed very quickly by avoiding the time consuming process of GTG-banding. The application of FISH to mouse cytogenetics would allow large scale molecular toxicology studies to be conducted on the effects of such environmental insults as potential carcinogens, mutagens and radiation. Progress has been hampered, however, as the Mus musculus karyotype consists of 40 acrocentric chromosomes of approximately the same size, making the recognition and separation of individual chromosomes very difficult. We now describe the successful production and application of chromosome-specific composite DNA probes for M. musculus chromosomes 2 and 8. Stable Robertsonian translocated chromosomes were isolated on a flow sorter and their DNA subsequently amplified by degenerate oligonucleotide primer (DOP) PCR. Small pools (300 copies) of each chromosome were denatured at 94 degrees C then annealed with the primer at 30 degrees C for 15 cycles. This was followed by 20 cycles at an annealing temperature of 62 degrees C. Additional amplification was performed at an annealing temperature of 62 degrees C. The chromosome-specific DNA was labeled with biotin 11-dUTP by nick translation and used for FISH. The usefulness of the technique for translocation detection is demonstrated by analyzing chromosome exchanges induced in mice irradiated with 137Cs gamma rays.

Comparative chromosome painting discloses homologous segments in distantly related mammals

“…This would correspond to the smallest trans-located segments which have been decteted in human metaphase spreads using whole chromosome paint probes. Besides DNA libraries established from flow sorted chromosomes of various species 13,14 ' 31,32 , microdissection libraries 7 ' 15,33 , probe contigs which span chromosome segments of interest and probes defining individual coding gene loci (type I anchor loci) 4 can be used in multicolour ZOO-FISH schemes. In this way the overall or local resolution of the comparative chromosome map from each species can be adjusted to the needs of each investigation.…”

The Importance of Age and Smoking in Evaluating Adverse Cytogenetic Effects of Exposure to Environmental Agents

“…When (11)(12)(13). The development of painting probes for mice (14)(15)(16)(17) has led to at least one experiment designed to provide quantitative estimates of the persistence of translocations (18). This persistence appears to be radiation dose-dependent, although more work must be done.…”

The development of painting probes for dual-color and multiple chromosome analysis in the mouse

“…has 40 similarly sized acrocentric chromosomes that appear as a series of poorly resolved peaks in a bivariate How karyotype. By using cells from mice bearing Robertsonian translocation chromosomes, we have overcome the obstacle of isolating pure chromosomes (Breneman et al, 1993). Amplification of DNA from flowsorted Robertsonian translocation chromosomes was accomSupported in part by grant CA5586I-01 from the National Institutes of Health.…”

“…How ever. painting of chromosomes from other species' cells has only recently become available because chromosomally pure DNA has been difficult to isolate (Dixon et al" 1992;Langford et al, 1992;Breneman et al" 1993;Miyashita et al, 1994). The common laboratory' mouse, Mus nntsculus.…”