The Zon Laboratory Guide to Positional Cloning in Zebrafish

CHAPTER 17

The Zon Laboratory Guide to Positional Cloning in Zebrafish Nathan Bahary,* Alan Davidson,*,{ David Ransom,* Jennifer Shepard,* Howard Stern,* Nikolaus Trede,* Yi Zhou,* Bruce Barut,{ and Leonard I. Zon*,{ *Division of Hematology/Oncology Children’s Hospital Boston Boston, Massachusetts, 02115 {

Howard Hughes Medical Institute Harvard Medical School Boston, Massachusetts, 02115

I. II. III. IV.

Introduction Mapping Strains Families and Genetic Markers Crosses for Line Maintenance and Mapping A. Choosing Grandparents and Parents for Better High-Resolution Mapping B. Microsatellite Markers—Agarose Scorable V. Preparation of the DNA VI. Mapping Genes A. Low-Resolution Mapping B. Intermediate-Resolution Mapping C. Fish Husbandry D. Tempo E. High-Resolution Mapping F. Three-Allele Systems Versus Four-Allele Systems G. Collecting Mutant Embryos H. Chromosomal Walking I. Screening the PAC or BAC Libraries J. New BAC Libraries K. Care of the BAC and PAC Libraries VII. Overgo Strategy for Rapidly Doing Chromosomal Walks and Positional Cloning

METHODS IN CELL BIOLOGY, VOL. 77 Copyright 2004, Elsevier Inc. All rights reserved. 0091-679X/04 $35.00

305