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(Frame, Stothard and Miles, unpublished data).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).