Quantification of transformation efficiency using a new method for clonal growth and selection of axenic Dictyostelium cells.
A new method for clonal growth of Dictyostelium axenic amoebae has been developed. Cells are plated in growth medium containing 1% ultra-low gelling temperature agarose. Cells grow normally in the agarose and form colonies up to several millimeters in diameter. When the colonies have grown to a sufficient size, they begin multicellular development. Pseudoplasmodia are formed, migrate to the surface of the agar, and then undergo fruiting body formation. Cells can be removed from the soft agarose colonies with a toothpick or by picking spores from the fruiting bodies. This method should be useful for drug, auxotrophic, and temperature selections where clonal maintenance of axenic colonies is important. This method has been used in combination with a selection for resistance to G418 to isolate independent colonies following DNA-mediated transformation. Several parameters in the calcium phosphate and electroporation transformation protocols have been optimized and the transformation frequency quantified. Independent transformed colonies are obtained at a frequency of 1 in 10(4) to 1 in 10(5) cells when integrating plasmids are introduced using calcium phosphate coprecipitation. The frequency is about tenfold higher when extrachromosomal shuttle vectors are introduced into cells.
Knecht
D A
DA
Department of Molecular and cell Biology, University of Connecticut, Storrs 06269.
Jung
J
J
Matthews
L
L
D000362:Agar; D015139:Blotting, Southern; D002130:Calcium Phosphates / Q000494:pharmacology; D002999:Clone Cells; D003470:Culture Media; D004271:DNA, Fungal / Q000032:analysis / Q000187:drug effects; D004023:Dictyostelium / Q000235:genetics / Q000254:growth & development*; D004560:Electricity; D005800:Genes, Fungal; D009154:Mutation; D014170:Transformation, Genetic
D002130:Calcium Phosphates; D003470:Culture Media; D004271:DNA, Fungal; C485828:alpha-tricalcium phosphate; C485830:tetracalcium phosphate; C485838:calcium phosphate, monobasic, anhydrous; D000362:Agar; C020243:calcium phosphate; C485829:calcium phosphate, dibasic, anhydrous