Gene trapping with GFP: the isolation of developmental mutants in the slime mold Polysphondylium.
In order to study how a cell mass undergoes a transition from one symmetry to another in the slime mold Polysphondylium, we developed a genetic screen in which mutant phenotype and gene expression can easily be visualized in the living organism. The screen combines restriction enzyme-mediated integration (REMI) [1,2] and green fluorescent protein (GFP) [3] expression. In REMI, a restriction enzyme is electroporated along with linearized vector into cells, thus determining the site of plasmid insertion and often increasing the integration frequency. A set of transforming plasmids carrying the GFP coding sequence in three reading frames was used for transformation. The plasmids were constructed so that GFP could be expressed only under control of a host promoter. Living transformants expressing GFP spatially and temporally could be rapidly identified in a very large background of non-expressing cells and fruiting bodies. The phenotypes of representative mutants range from cells that cannot aggregate and initiate cell-cell interactions, through mutant fruiting bodies, to apparently wild-type fruiting bodies expressing GFP in all or a subpopulation of cells. The ability to screen mutant living cells and tissues for GFP expression is rapid and effective and likely to have application in many transformable systems where screening by gene and promoter trapping is essential for understanding temporal and spatial gene regulation.
Fey
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Department of Molecular Biology, Princeton University, New Jersey 08544, USA.
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D003001:Cloning, Molecular / Q000379:methods*; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000235:genetics*; D009154:Mutation; D009235:Myxomycetes / Q000235:genetics* / Q000254:growth & development*; D010641:Phenotype; D010957:Plasmids; D015183:Restriction Mapping; D014170:Transformation, Genetic
D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins