CROSSING EXPERIMENTS WITH EPISOMAL TRANSFORMANTS

Development of genetic transformation methods has provided a means of introducing drug-resistant markers to trypanosome populations. Transfection of neomycin phosphotransferase confers resistance to neomycin (G418) and of hygromycin phosphotransferase confers resistance to hygromycin. Putative parental T. cruzi populations that have been genetically transformed to carry different single drug-resistant markers enable selection of dual drug-resistant progeny after crossing experiments.

Ideally, parents for such experimental crosses should be T. cruzi isolates from a single locality (sympatric isolates) and also from what is considered to be a single type of transmission cycle. Accordingly, we have undertaken some experimental crosses with the putative parental T. cruzi isolates described by Carrasco et al. (1996), both assigned to Z1, and occurring sympatrically in the Amazon basin. A detailed account of these experiments will be published elsewhere (Frame, Stothard and Miles, unpublished data). Briefly, we have used a plasmid carrying neomycin resistance and a second plasmid carrying hygromycin resistance to confer drug resistance, stable for sustained passage, as described by Kelly (1997). Following copassage of putative parental T. cruzi populations carrying single drug-resistant markers, dual drug-resistant populations were selected by growth in medium containing predetermined optimal combinations of both neomycin and hygromycin. Appropriate controls included single drug-resistant transformants and mock transfected T. cruzi populations. Biological clones of T. cruzi were prepared from dual drug-resistant populations.

A multiplex PCR was designed to examine the basis of dual drug resistance in the biological clones that were the product of crossing experiments. Two oligonucleotide primer pairs were made, one which gave a specific amplification product size to detect neomycin resistance and a second which gave a PCR product of a different size to detect hygromycin resistance. As the primer pairs do not cross react both can be included in a single (multiplex) PCR reaction. The size difference of the products enables rapid, simple determination of the presence of neomycin resistance, hygromycin resistance, or both forms of resistance in very small numbers of organisms. A particular advantage of the multiplex PCR is thus the rapid screening of large numbers of progeny populations without growing them in bulk. This multiplex PCR has confirmed the carriage of both episomal plasmids in dual drug-resistant T. cruzi populations obtained from crossing experiments. Acquisition of both episomal constructs in dual drug-resistant biological clones is also confirmed by Southern hybridisation. Furthermore, preliminary karyotype analysis suggests that recombination of parental genotypes is not confined to the extranuclear genome (Frame, Stothard & Miles, unpublished data).