Colony polymerase chain reaction of stably transfected trypanosoma cruzi grown on solid medium.
Tools for the genetic manipulation of Trypanosoma cruzi are largely unavailable, although several vectors for transfection of epimastigotes and expression of foreign or recombinant genes have been developed. We have previously constructed several plasmid vectors in which recombinant genes are expressed in T. cruzi using the rRNA promoter. In this report, we demonstrate that one of these vectors can simultaneously mediate expression of neomycin phosphotransferase and green fluorescent protein when used to stably transfect cultured epimastigotes. These stably transfected epimastigotes can be selected and cloned as unique colonies on solid medium. We describe a simple colony PCR approach to the screening of these T. cruzi colonies for relevant genes. Thus, the methodologies outlined herein provide important new tools for the genetic dissection of this important parasite.
Santos
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WG
Department of Microbiology and Immunology, Medical College of Virginia Campus, Virginia Commonwealth University, Richmond, VA, 23298-0678, USA.buck@hsc.vcu.edu
Metcheva
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I
Buck
G A
GA
D000818:Animals; D003470:Culture Media; D017931:DNA Primers; D015870:Gene Expression; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000235:genetics*; D019868:Kanamycin Kinase / Q000235:genetics / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods*; D014162:Transfection / Q000379:methods*; D014349:Trypanosoma cruzi / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development
D003470:Culture Media; D017931:DNA Primers; D019868:Kanamycin Kinase