The Isolation of Molecular Markers

If a positional cloning exercise is to have a realistic chance to succeed, it requires the existence of enough molecular markers for chromosome walks to be initiated. In many cases such markers are not available and strategies are devised to generate them. The use of somatic cell hybrids and chromosome microdissection have been the only two approaches successfully applied to generate molecular markers for mouse chromosomes. Although a plethora of mouse microsatellite markers is now available, there are still regions which are not well represented on the microsatellite map (Dietrich at a!., 1992 and 1994). Therefore, it is still necessary to employ these and other strategies in order to ensure a complete coverage of the mouse genome in terms of molecular markers.

4.2.1 Somatic Cell Hybrids

Somatic cell hybrids are generated by the fusion of cells (usually fibroblasts) from two species to produce hybrid cell lines which contain chromosomes from both species (reviewed by Abbott and Povey, 1995). These lines retain all the chromosomes from one of the parental lines but lose one or more chromosomes from the other parent. The result from a fusion experiment between a hamster and a mouse cell line is a panel of interspecific hybrid cell lines, each containing a different set of mouse chromosomes in the presence of all the hamster ones. Ideally, one is seeking to identify a hybrid line that will contain only the mouse chromosome of interest (mono-chromosomal hybrid) which can then be used as a source for

The problem often encountered in mouse-hamster hybrid cell lines is the considerable fragmentation of the mouse chromosomes. This means that the exclusion of one marker from a cell line does not imply the absence of nearby markers or the whole of the chromosome. Therefore, the characterisation of these lines requires a representative number of molecular markers for each chromosome. Until recently, the molecular markers were not available at sufficient density across the genome and certain regions were poorly represented. Normal mouse chromosomes are very difficult to distinguish cytogenetically because they show a continuous gradation in size, so this option is not available either. For these reasons the use of somatic cell hybrids either in gene mapping or as a resource for chromosome-specific markers has been rather limited.

Somatic cell hybrids generated by whole-cell fusion may not yield lines that contain the desired chromosome. Microcell-mediated gene transfer (MMGT) was developed to allow the transfer of single chromosomes into a recipient cell line (Fournier and Ruddle, 1977). Briefly, the donor cells are blocked in mitosis by treatment with colcemid. The nuclear membrane gradually reforms around single or small groups of chromosomes to form multiple micronuclei. After treatment with cytochalasin B the nuclear membrane is removed. Centrifugation produces individual microcells (single micronuclei encapsulated in the plasma membrane). These can be fused with recipient cells to produce a hybrid cell line with a single (theoretically) chromosome from the donor species.

The markers generated from mono-chromosomal hybrid lines could make a significant contribution towards a specific positional cloning exercise, provided sufficient resources are available to yield as many markers as possible. The alternative to mono-chromosomal somatic cell hybrids is the use of irradiation-fusion gene transfer (IFGT) hybrids which carry only a small region of a chromosome (Goss and Harris, 1975). Lines that contain the region of interest could then be used as a source of molecular markers. IFGT mouse-hamster hybrid lines are generated by irradiating (X or y-rays) of a monochromosomal hybrid line followed by fusion to a hamster parent to rescue single fragments of the mouse chromosome in different, newly formed, hybrid lines (Goss and Harris, 1975). The characterisation of IFGT hybrid lines requires the existence of sufficient region-specific markers to define the boundaries of the rescued fragment. Such hybrids can provide an excellent source for region-specific markers either through the use of IRS-PCR (Herman et al., 1991) or by constructing genomic libraries to isolate mouse-specific clones. IFGT hybrid lines have been used extensively in human genetics to obtain genetic markers (Burright ef a/., 1991; Ragoussis eta!., 1991; Cotter ef a/; 1990)

Mouse-hamster hybrid lines have been used to generate molecular markers for mouse chromosomes in two main strategies. Both approaches exploit the presence of three predominant families of dispersed repetitive DNA (reviewed by

Hastie, 1989). One family includes the Limd (or L1) element, abbreviated LINES for long interspersed repeats. The full-size of the repeat is approximately 7 kb. Most repeats are truncated at the 5’ end, the most frequent form of LINES (Voliva et al.,

1983). This family covers about 5% of the mouse genome. The other two families are the B1 and B2 short interspersed repeats (SINES) with repeat lengths of about 130 and 190 bp respectively. The SINES form about 2% of the mouse genome. Cytogenetic analysis of the mouse genome suggests that LINES are predominantly found in late-replicating G (Geisma) bands, whereas the SINES are mostly present in R {reverse G bands) bands (Boyle et a!., 1990). In spite of the very similar architecture between the mouse and hamster SINES and LINES, there is sufficient sequence divergence for them to be used as species-specific tags (reviewed by Herman eta!., 1992).

The first strategy involves the construction of a genomic or subgenomic library which is then screened with either a composite probe, derived by radiolabelling mouse genomic DNA (Hochgeschwender et a!., 1989), or pooled individual clones which contain unidentified repetitive elements of the mouse genome, excluding members of the satellite and B1 families (Hoglund, et a!., 1992). A more detailed account of the former approach is given in chapter 2.

The second strategy is less laborious and employs the PCR. It takes advantage of the very high sequence conservation among individual members of single families within a species. Primers are designed from a consensus, mouse- specific, region from either end of the repeat and used to amplify DNA from mouse- hamster somatic cell hybrids (Cox et a!., 1991; Irving and Brown, 1991; Simmler et a!., 1991). If two repeats evolved adjacent to each other but in opposite orientation then a single primer will prime the amplification of the intervening region, provided that this region can be amplified by the PCR. The end-result is a pool of mouse specific fragments which can be recovered and used as a source of markers for the chromosome(s) involved (Herman et a!., 1991; Cox et a!., 1991; Irving and Brown, 1991; Simmler et a!., 1991). This approach, known as interspersed repetitive sequence PCR (IRS-PCR), was modified to target regions which do not contain two adjacent repeats but only one in order to obtain a more representative set of markers (M un roe et a!., 1994).

4.2.2 Chromosome Microdissection

In 1981 it was demonstrated that single DNA molecules could be recovered from Drosophila polytene chromosome bands using a fine-tip glass needle attached to a micromanipulator (Scalenghe et al., 1981; reviewed by Kao, 1993). After an initial treatment to remove impurities, the fragments were cloned to generate a region-specific library. Metaphase-chromosome microdissection was first used in the mouse to generate clones from the t-complex region on mouse chromosome 17

(Rohme et al ,1984). In order to distinguish chromosome 17, a strain carrying a Robertsonian translocation involving chromosomes 8 and 17 was used, chromosome 17 being the short arm of the only metacentric chromosome in chromosome spreads from this strain. The clones that were obtained from that effort provided the starting material which led to the isolation of the Brachyury gene (Herrmann at a!., 1990). Because only minute amounts of DNA can be recovered by chromosome microdissection, the cloning efficiency is very low. Protocols have been adopted which make use of the PCR for an initial amplification of the recovered DNA prior to cloning (Ludecke at a!., 1989). This has improved the cloning efficiency and the coverage of the target region in the resulting libraries. The clones derived by this approach can be used in genetic analysis or to screen YAC libraries (Vidal at a/; 1992). The use of chromosome microdissection is rather limited because it is technically very demanding and not very successful.