Chapter 16 Construction and Characterization of Zebrafish Whole Genome Radiation Hybrids

Abstract

This chapter describes the construction and characterization of zebrafish whole genome radation hybrids. Zebrafish whole genome radiation hybrids are produced by fusing irradiated zebrafish cells to a hamster cell line that is hypoxanthine phosphoribosyl transferase (HPRT) or thymidine kinase (TK) deficient. The irradiation procedure randomly breaks the zebrafish genome, and the DNA fragments generated are rescued via the fusion process. Surviving cells are grown in hypoxanthine-aminopterinethymidine (HAT) medium, which selects for hybrids containing the zebrafish HPRT gene or TK gene. After picking the surviving hybrid clones and DNA extraction, the panel of hybrids is tested by polymerase chain reaction (PCR) for retention of genomic fragments by typing several microsatellite markers across the genome. For large-scale mapping or positional cloning projects, statistical programs are available to calculate map order and distances based on PCR typing of many markers across the hybrid panel. The advantages of whole genome radiation hybrids are (1) the ability to map any marker as long as it differs between rodent and zebrafish and (2) tailoring panels to different mapping resolutions depending on the application by adjusting the radiation dose.