A novel GFP approach for the analysis of genetic exchange in trypanosomes allowing the in situ detection of mating events

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Abstract
Trypanosoma brucei undergoes genetic exchange in its insect vector by an unknown mechanism. To visualize the production of hybrids in the fly, a tetracycline (Tet)-inducible expression system was adapted. One parental trypanosome clone was transfected with the gene encoding Green Fluorescent Protein (GFP) under control of the Tet repressor in trans; transfection with these constructs also introduced genes for resistance to hygromycin and phleomycin, respectively. An experimental cross with a second parental clone carrying a gene for geneticin resistance produced fluorescent hybrids with both hygromycin and geneticin resistance. These results are consistent with the meiotic segregation and reassortment of the GFP and repressor genes. Fluorescent hybrids were visible in the salivary glands of the fly, but not the midgut, confirming that genetic exchange occurs among the trypanosome life cycle stages present in (or possibly en route to) the salivary glands. In conclusion, the experimental design has successfully produced fluorescent hybrids which can be observed directly in the salivary glands of the fly, and it has been shown that the recombinant genotypes were most probably the result of meiosis.