Variables controlling the expression level of exogenous genes in Dictyostelium.
Ectopic expression of genes from recombinant plasmids is commonly used to study gene function. In Dictyostelium, three drug resistance cassettes are commonly used as selectable markers in vectors. We report here a comparative study of the expression of green fluorescent protein (GFP) gene from vectors containing each of the drug-resistant cassettes. The expression was highest in cells transformed with the vectors containing the neomycin-resistant cassette (pDNeoGFP), followed by the hygromycin-resistant cassette (pDHygGFP) and the blasticidin-resistant cassette (pDBsrGFP). The level of GFP expression was directly related to the copy number of the vector in transformants. In turn, the copy number of the vector depended on the drug resistance cassette as well as the concentration of the drug used in selection. In general, cells with higher copy numbers could be selected by a higher drug concentration. The expression of GFP was also affected by the method of transformation. For pDHygGFP, expression of GFP was much higher in cells transformed by electroporation than those transformed by calcium phosphate coprecipitation. However, only a slight difference was observed for pDNeoGFP or pDBsrGFP.
Pang
K M
KM
Department of Molecular and Cell Biology, University of Connecticut, Storrs, Connecticut 06269, USA.
Lynes
M A
MA
Knecht
D A
DA
D000818:Animals; D002934:Cinnamates; D004023:Dictyostelium / Q000235:genetics*; D004351:Drug Resistance / Q000235:genetics; D015870:Gene Expression; D005819:Genetic Markers; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006921:Hygromycin B / Q000031:analogs & derivatives / Q000494:pharmacology; D008164:Luminescent Proteins / Q000235:genetics; D009355:Neomycin / Q000494:pharmacology; D009705:Nucleosides / Q000494:pharmacology; D010957:Plasmids / Q000235:genetics; D014170:Transformation, Genetic
D002934:Cinnamates; D005819:Genetic Markers; D008164:Luminescent Proteins; D009705:Nucleosides; D049452:Green Fluorescent Proteins; D006921:Hygromycin B; C026273:hygromycin A; C004500:blasticidin S; D009355:Neomycin