Transgenic animal models are essential experimental tools for unraveling gene function in normal development and disease. An ideal transgene recapitulates the endogenous gene expression pattern. To this end, it should encompass all the regulatory sequences required for recapitulating endogenous expression. Since these regulatory sequences are often far apart, such transgenic constructs frequently need to be a few hundred kilo bases (kb) of DNA in length. Large fragments (100-200 kb) of the genomes of most animal model systems have been subcloned into bacterial artificial chromosomes (BACs). Such BACs can be modified through recombineering (recombinant genetic engineering). However, techniques that facilitate efficient BAC transgenesis are lacking. The goal of this proposal is to develop a technique for efficient insertion of BAC constructs into the genome of the model organism zebrafish. Past efforts show that injection of naked BAC DNA or co-injection of Tol2 transposase with BAC DNA flanked by Tol2 sites into fertilized zebrafish eggs results in comparable transgenesis rates of 0.5-5%. Here, we propose to test a modified Tol2-mediated BAC transgenesis approach. Specifically, we intend to (1) develop universal tools for incorporating transposase sites into any BAC, (2) test the versatility of our modified Tol2-mediated BAC transgenesis approach for use with BACs harboring small and large DNA fragments and, as a proof of principle, generate Cre reporter lines.

Public Health Relevance

Transgenic zebrafish models are essential for our understanding of gene function in normal development and disease. While transgenesis of small DNA constructs is a standard technique in zebrafish, the integration of 100 kilo bases or more into the zebrafish genome is hampered by its low efficiency of transgenesis. In this proposal, we intend to develop a technique for efficient integration of large transgenes into the zebrafish genome.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Small Research Grants (R03)
Project #
1R03HD070866-01
Application #
8222984
Study Section
Pediatrics Subcommittee (CHHD)
Program Officer
Coulombe, James N
Project Start
2012-04-01
Project End
2014-03-31
Budget Start
2012-04-01
Budget End
2013-03-31
Support Year
1
Fiscal Year
2012
Total Cost
$84,500
Indirect Cost
$34,500
Name
New York University
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
121911077
City
New York
State
NY
Country
United States
Zip Code
10016