The goal of the proposed work is to develop new methods for precisely altering the genome of zebrafish efficiently. The technology to be developed here will dramatically expand and likely revolutionize the kinds of experiments that can be performed and the kinds of questions that can be asked with the zebrafish. Our approach uses homologous recombination to replace specific sequences in the host genome with introduced exogenous sequences. The immediate aims are: 1) to demonstrate targeted genes can be altered by homologous recombination in somatic tissues, 2) to optimize conditions to accomplish homologous recombination utilizing single strand or double strand donor DNA molecules, 3) to measure the occurrence of genome integration of donor DNA sequences at unintended loci by non-homologous recombination mechanisms, and 4) to demonstrate that heritable alterations can be produced and recovered in subsequent generations. New technologies have recently been developed that permit the very efficient induction of double strand DNA breaks at specific loci using engineered nucleases. The chromosomal DNA breaks stimulate recombination at the site of the lesion and have been used to initiate gene targeting in fruit flies and cultured mammalian cells. Our preliminary results indicate that simultaneous introduction of target-specific nucleases and single strand donor DNA molecules promotes homologous recombination in the somatic tissues of zebrafish embryos resulting in designed sequence changes at a targeted host gene. We anticipate the proposed experiments will identify conditions needed for the routine manipulation of the zebrafish genome.

Public Health Relevance

The proposed research will develop new, innovative genetic methods for precise manipulation of the zebrafish genome by homologous recombination. Over the last decade the zebrafish has emerged as a major model organism used for elucidating genetic, developmental, physiological, and behavioral processes conserved among vertebrates. The new technology to be developed here will dramatically expand (likely revolutionize) the kinds of experiments that can be performed and the kinds of questions that can be asked with the zebrafish.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Exploratory/Developmental Grants (R21)
Project #
5R21HD073847-02
Application #
8534228
Study Section
Development - 1 Study Section (DEV1)
Program Officer
Coulombe, James N
Project Start
2012-08-20
Project End
2014-07-31
Budget Start
2013-08-01
Budget End
2014-07-31
Support Year
2
Fiscal Year
2013
Total Cost
$176,751
Indirect Cost
$58,126
Name
University of Utah
Department
Genetics
Type
Schools of Medicine
DUNS #
009095365
City
Salt Lake City
State
UT
Country
United States
Zip Code
84112
Grunwald, David Jonah (2013) A revolution coming to a classic model organism. Nat Methods 10:303, 305-6
Hu, Ruozhen; Wallace, Jared; Dahlem, Timothy J et al. (2013) Targeting human microRNA genes using engineered Tal-effector nucleases (TALENs). PLoS One 8:e63074