The zebrafish possesses characteristics that make it an ideal model for genetic studies of vertebrate development and human disease. Although large-scale forward mutagenesis screens have been successfully applied to the zebrafish to identify genes that regulate early development, methods are not available for targeted gene inactivation by insertional mutagenesis. The goal of this research is to develop gene-targeting methods using zebrafish embryonic stem (ES) cell lines. To accomplish this goal, the existing zebrafish ES cell culture system will be optimized for use in the production of knockout lines of fish. Conditions will be established that support the optimal growth and survival of germ-line competent ES cells in culture and methods will be developed to improve the efficiency of homologous recombinant colony isolation. To demonstrate the feasibility of this gene targeting approach, the ES cell lines will be utilized to target the inactivation of a gene that is important for normal development, producing a knockout mutant possessing an obvious and well-characterized phenotype.
The specific aims of this research are: 1) determine the frequency of germ-line chimera production using late-passage zebrafish ES cell lines; 2) establish clonally-derived ES cell lines that produce germ-line chimeras at a high frequency; 3) derive a feeder cell line from zebrafish spleen that is more effective than the currently used trout spleen cell line at maintaining germ-line competency of the zebrafish ES cells; 4) identify the most efficient method to isolate colonies of homologous recombinants in the ES cell cultures; 5) use ES cell cultures to target the inactivation of the no tail gene to demonstrate the feasibility of this gene-targeting approach. The ES cell-based gene targeting approach developed from this work will complement other genetic methods currently applied to zebrafish and increase the value of this organism as a model for the study of genes important in human development and disease.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM069384-01
Application #
6684528
Study Section
Special Emphasis Panel (ZRG1-CDF-5 (50))
Program Officer
Greenberg, Judith H
Project Start
2003-08-01
Project End
2006-07-31
Budget Start
2003-08-01
Budget End
2004-07-31
Support Year
1
Fiscal Year
2003
Total Cost
$326,246
Indirect Cost
Name
Purdue University
Department
Veterinary Sciences
Type
Schools of Earth Sciences/Natur
DUNS #
072051394
City
West Lafayette
State
IN
Country
United States
Zip Code
47907
Wong, Ten-Tsao; Collodi, Paul (2013) Dorsomorphin promotes survival and germline competence of zebrafish spermatogonial stem cells in culture. PLoS One 8:e71332
Wong, Ten-Tsao; Collodi, Paul (2013) Effects of specific and prolonged expression of zebrafish growth factors, Fgf2 and Lif in primordial germ cells in vivo. Biochem Biophys Res Commun 430:347-51
Wong, Ten-Tsao; Tesfamichael, Abraham; Collodi, Paul (2013) Production of zebrafish offspring from cultured female germline stem cells. PLoS One 8:e62660
Wong, Ten-Tsao; Saito, Taiju; Crodian, Jennifer et al. (2011) Zebrafish germline chimeras produced by transplantation of ovarian germ cells into sterile host larvae. Biol Reprod 84:1190-7
Liu, Weiyi; Collodi, Paul (2010) Zebrafish dead end possesses ATPase activity that is required for primordial germ cell development. FASEB J 24:2641-50
Liu, Weiyi; Guan, Yihong; Collodi, Paul (2010) A zebrafish cell culture assay for the identification of microRNA targets. Zebrafish 7:343-8
Xing, J G; El-Sweisi, W; Lee, L E J et al. (2009) Development of a zebrafish spleen cell line, ZSSJ, and its growth arrest by gamma irradiation and capacity to act as feeder cells. In Vitro Cell Dev Biol Anim 45:163-74
Fan, Lianchun; Moon, Jesung; Wong, Ten-Tsao et al. (2008) Zebrafish primordial germ cell cultures derived from vasa::RFP transgenic embryos. Stem Cells Dev 17:585-97
Barnes, David W; Parton, Angela; Tomana, Mitsuru et al. (2008) Stem cells from cartilaginous and bony fish. Methods Cell Biol 86:343-67
Xing, Jerry G; Lee, Lucy E J; Fan, Lianchun et al. (2008) Initiation of a zebrafish blastula cell line on rainbow trout stromal cells and subsequent development under feeder-free conditions into a cell line, ZEB2J. Zebrafish 5:49-63

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