Transposon-Based Platform for Insertional Mutagenesis and Genome Engineering Abstract: The fastest, most comprehensive and cost-effective way to study human gene function is through their counterparts in model systems such as zebrafish. Two key characteristics of any gene are expression pattern and loss of function phenotype. We propose to develop tools that for the first time in a vertebrate simultaneously assay both of these characteristics. Our vectors will report gene expression patterns using Gal4/UAS while conditionally mutating genes in zebrafish. Gal4/UAS will enable investigators to use our gene traps as driver lines to express other proteins of interest in specific tissues. Gene trap cassettes will be flanked by a combination of site-specific recombinase recognition sites for regulation of mutagenicity. During the process of improving and testing our vectors we will generate, propagate and make available 150 zebrafish gene trap lines. To complement these gene trap lines, we will produce several lines with tissue-specific expression of Cre recombinase for spatial regulation of mutagenicity and use them to revert integrations into genes essential for viability. These tissue-specific Cre lines will be also useful for genetic lineage tracing. Since our gene traps will contain recognition sites for site-specific recombinases, the 120 gene trap lines will constitute a library of recombinase recognition sites distributed throughout the zebrafish genome. This will allow us to carry out proof of principle experiments for use of site-specific recombinases to generate large inversions and deletions, and enable other researchers to generate zebrafish "designer chromosomes" of interest.

Public Health Relevance

Genetics plays a substantial role in many human diseases. This project will develop tools to greatly enhance our ability to study gene function in a vertebrate model system- the zebrafish.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Research Project (R01)
Project #
5R01HD061749-03
Application #
8280376
Study Section
Special Emphasis Panel (ZRG1-BDA-L (50))
Program Officer
Coulombe, James N
Project Start
2010-08-01
Project End
2015-05-31
Budget Start
2012-06-01
Budget End
2013-05-31
Support Year
3
Fiscal Year
2012
Total Cost
$304,776
Indirect Cost
$105,576
Name
Temple University
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
057123192
City
Philadelphia
State
PA
Country
United States
Zip Code
19122
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Roberts, Jennifer Anne; Miguel-Escalada, Irene; Slovik, Katherine Joan et al. (2014) Targeted transgene integration overcomes variability of position effects in zebrafish. Development 141:715-24
Balciuniene, Jorune; Nagelberg, Danielle; Walsh, Kathleen T et al. (2013) Efficient disruption of Zebrafish genes using a Gal4-containing gene trap. BMC Genomics 14:619
Balciuniene, Jorune; Balciunas, Darius (2013) Gene trapping using gal4 in zebrafish. J Vis Exp :e50113