A transformation vector for stage-specific expression of heterologous genes in Trypanosoma cruzi epimastigotes.
To express heterologous genes in a stage-specific manner, we constructed a transformation vector for Trypanosoma cruzi containing a selection gene (hyg) and a reporter gene (luc) flanked by sequences of the multicopy 1f8 gene arranged so as to provide a trans-splicing acceptor site to hyg and a putative polyadenylation signal to luc. The intergenic region of the T. cruzi genes 294 and KAP was placed between hyg and luc, contributing the polyadenylation signal of 294 to hyg and the KAP trans-splicing acceptor site to luc. Transformation was carried out by electroporation, and transformed epimastigotes were selected in medium containing hygromycin B. Through double homologous recombination of the 1f8 sequences with their chromosomal counterparts, the construction is inserted into the 1f8 locus, substituting probably one and no more than a few copies of the 1f8 gene without having apparent deleterious effects on the parasite. cDNA analysis demonstrated that the introduced signals were correctly processed, resulting in translatable hyg and luc mRNAs. Whereas epimastigotes express luciferase, no expression is found in the trypomastigote stage.
Thomas
M C
MC
Instítuto de Parasitología y Biomedicina, CSIC, Granada, Spain.
González
A
A
D000818:Animals; D030801:Animals, Genetically Modified; D001483:Base Sequence; D017931:DNA Primers; D018076:DNA, Complementary / Q000032:analysis; D018274:Electroporation; D017930:Genes, Reporter; D005822:Genetic Vectors; D008156:Luciferases / Q000096:biosynthesis / Q000235:genetics; D008969:Molecular Sequence Data; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000096:biosynthesis / Q000235:genetics; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis*; D011995:Recombination, Genetic; D014162:Transfection; D014349:Trypanosoma cruzi / Q000235:genetics / Q000502:physiology*
D017931:DNA Primers; D018076:DNA, Complementary; D011993:Recombinant Fusion Proteins; D008156:Luciferases; D017853:Phosphotransferases (Alcohol Group Acceptor); C063242:hygromycin-B kinase