BRIEF REPORT |
Microdissection-derived Murine Mcb Probes from Somatic Cell Hybrids
Institute of Human Genetics and Anthropology, Jena, Germany (VT,CK,UC,KM,SM,TL); Institute of Cytology and Genetics, Novosibirsk, Russia (VT); and Unité de Génétique Moléculaire Murine, Institut Pasteur, Paris, France (PA)
Correspondence to: Dr. Thomas Liehr, Institut für Humangenetik, Postfach D-07740, Jena, Germany. E-mail: i8lith{at}mti.uni-jena.de
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Summary |
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Key Words: multicolor banding murine chromosomes somatic cell hybrids fluorescence in situ hybridization
ALTHOUGH THE MOUSE (Mus musculus) is "the most accessible mammalian model" (Rangarajan and Weinberg 2003) for cytogenetic studies, this species is not very popular. The mouse carries 20 chromosome pairs, which are all acrocentric and uneasy to distinguish after GTG-banding. Thus, murine chromosomal rearrangements can only be characterized in detail using molecular cytogenetic approaches, and the introduction of fluorescence in situ hybridization (FISH) using murine whole chromosome painting (wcp) probes (Liyanage et al. 1996
; Schröck et al. 1996
) enabled a more detailed analysis of chromosomal rearrangements in a variety of cell lines (for overview see Liehr 2005
). However, FISH methods using wcp probes reach their limits when exact localization of chromosomal breakpoints is required or when intrachromosomal rearrangements such as small interstitial deletions or duplications and inversions occur. FISH-banding techniques are suitable for the characterization of all kinds of chromosomal rearrangements and marker chromosomes (Liehr et al. 2002a
; Liehr 2005
). As no multicolor FISH-banding technique for the mouse has been available up to now, the easily applicable and well-established multicolor-banding technique called MCB, or m-bands when using human probes (for overview see Liehr 2005
), was adapted for Mus musculus and called mcb.
Here we present for the first time the establishment of murine mcb-probe sets, as well as the establishment of mcb probes from somatic cell hybrids that was not previously reported.
The multicolor-banding technique is based on overlapping region-specific partial chromosome paints generated by glass-needle based microdissection (Chudoba et al. 1999; Liehr et al. 2002b
). Each of the probes is based on 1520 chromosomal fragments and the isolated DNA is amplified by degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR) (Telenius et al. 1992
). Between four and eight microdissection libraries were created per mouse chromosome. The chromosomal location of the partial chromosome painting probes (pcps) as shown in Figure 1 was confirmed by reverse painting to normal murine chromosomes. Four to five different fluorochromes are used to label the pcps: SpectrumOrange, Fluorescein, TexasRed, Cyanine 5 (i.e., Cy5 coupled to avidin for detection of biotinylated probes) and diethylaminocoumarine (DEAC). Probe labeling was done by DOP-PCR again. All technical details of MCB/mcb are described in Liehr et al. (2002b)
.
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In summary, the creation of mcb probes from somatic cell hybrids is a very elegant approach that could be applied to banding in all species that have cytogenetically hardly distinguishable chromosomes.
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Acknowledgments |
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Footnotes |
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Literature Cited |
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