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  • Title: Stable transformation of Trypanosoma cruzi: inactivation of the PUB12.5 polyubiquitin gene by targeted gene disruption.
    Author: Hariharan S, Ajioka J, Swindle J.
    Journal: Mol Biochem Parasitol; 1993 Jan; 57(1):15-30. PubMed ID: 8381205.
    Abstract:
    Analysis of gene expression in Trypanosoma cruzi has been impeded by the lack of efficient, stable, DNA-mediated transfection systems. We describe here the establishment of such a system for T. cruzi. Stable transformants were isolated following integration of the circular transforming plasmid into the chromosome by homologous recombination. Mutants with a disrupted PUB12.5 polyubiquitin gene, resulting from targeted integration of the plasmid vector, have been isolated. A mutant harboring the disrupted PUB12.5 gene lacks the intact PUB12.5 mRNA as well as transcripts corresponding to the truncated gene. Genomic Southern-blot analysis indicates that the inserted plasmid is tandemly repeated in each of the clones analyzed. A secondary recombination event in one clone resulted in a deletion within the 2.65 calmodulin-ubiquitin locus, encompassing the sequence from the CalA2 calmodulin gene to the PUB12.5 polyubiquitin gene.
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