Student Research Project
Identification and characterization of microsatellite markers in the rabbit genome

Students
Vinay Harpalani
Major: Biological Sciences
Future Plans: M.D./Ph.D. program

Vasudha Bhide
Major: Biological Sciences
Future Plans: Medical school

Professor
Patricia A. DeLeon, Professor, School of Life and Health Sciences, University of Delaware, Newark

The establishment of genetic marker maps is a prerequisite for genome analysis. In mammalian species, microsatellites have become the preferred genetic markers for establishing such maps. Microsatellites are dinucleotide repeats, such as GTGTGTGT..., which are scattered throughout the genomes of mammalian species. The length and pattern of these repeats are highly variable among individuals, and this variation, known as polymorphism, makes microsatellites very useful as genetic markers. Microsatellites have been characterized in the human, mouse, bovine, and porcine genomes but to date have not been studied in the rabbit (Oryctolagus cuniculus). One of my current student research projects involves the identification and characterization of microsatellite markers in the rabbit. Characterization and mapping of rabbit microsatellites will allow us to create a low resolution genetic map that will enhance comparative mapping between humans and rabbits. This could render the rabbit a more useful model for studying genetic diseases.

To identify and characterize microsatellites in the rabbit, we have used a variety of molecular and cytogenetic techniques. We isolated 40 Kb segments of DNA (cosmids) containing microsatellites by screening a rabbit genomic library with a radioactively labeled (GT)8 probe. We have mapped several cosmids onto specific rabbit chromosomes by fluorescence in situ hybridization (FISH). We use propidium iodide/DAPI, counter-staining for chromosome banding and thus more specific regional localization of the microsatellite-containing cosmids.

In addition to mapping, we have sequenced several cosmids using the automated dye terminator method. To accomplish this, degenerate primers containing (GT)8 and (TG)8 repeats are used to obtain up to 400 base pairs of sequence. From this sequence, flanking primers are designed to obtain the microsatellite sequence. In the future, we will use polymerase chain reaction (PCR) to identify different alleles of the mirosatellites in both outbred and inbred rabbits. We will also analyze the cosmid sequences to search for homologies in the gene database.

Our initial results have already begun to show similarities between rabbits and humans. One of the cosmids that maps to the centromeric region of chromosome 5 revealed the existence of satellite DNA as is found in human chromosome 16. This finding is most interesting due to the similarity in size, morphology, and centromeric banding of rabbit chromosome 5 and human chromosome 16. It is also consistent with an earlier gene assignment common to the proximal region of rabbit 5q and human 16q, producing strong evidence for homology between these two chromosomes. We have obtained preliminary microsatellite sequences that will be analyzed in the future.

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