Zebrafish ES cell lines for targeted mutagenesis
Principal Investigator: Paul Collodi
Affiliation: Purdue University
Abstract: 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.
Funding Period: 2003-08-01 - 2006-07-31
more information: NIH RePORT
- Zebrafish embryonic stem cellsLianchun Fan
Department of Animal Sciences, Purdue University, West Lafayette, Indiana, USA
Methods Enzymol 418:64-77. 2006..Two strategies are described for the efficient isolation of homologous recombinants using a visual marker screen and positive-negative selection...
- Initiation of a zebrafish blastula cell line on rainbow trout stromal cells and subsequent development under feeder-free conditions into a cell line, ZEB2JJerry G Xing
Department of Biology, University of Waterloo, Waterloo, Ontario, Canada
Zebrafish 5:49-63. 2008..ZEB2J should be useful for optimizing culture conditions for zebrafish embryonic stem cells...
- Stem cells from cartilaginous and bony fishDavid W Barnes
Mount Desert Island Biological Laboratory, Salisbury Cove, Maine, USA
Methods Cell Biol 86:343-67. 2008
- Zebrafish primordial germ cell cultures derived from vasa::RFP transgenic embryosLianchun Fan
Eli Lilly and Company, Indianapolis, IN 46221, USA
Stem Cells Dev 17:585-97. 2008....
- Development of a zebrafish spleen cell line, ZSSJ, and its growth arrest by gamma irradiation and capacity to act as feeder cellsJ G Xing
Department of Biology, University of Waterloo, 200 University Ave, Waterloo, ON, Canada N2L 3G1
In Vitro Cell Dev Biol Anim 45:163-74. 2009..In cocultures, nongrowth-arrested ZSSJ stimulated ZEB2J proliferation better than growth-arrested ZSSJ and better than RTS34st. ZSSJ should be useful as a feeder cell line for zebrafish ES cell cultures...