Bottlenecks and the Maintenance of Minor Genotypes during the Life Cycle of Trypanosoma brucei (original) (raw)
Figure 2
Cloning procedure and generation of tagged trypanosomes.
A. Cloned procyclic trypanosomes were passaged through a tsetse fly and a mouse and bloodstream forms were triggered to differentiate to procyclic forms in vitro. The plasmids piTag 1–8 were then transfected separately into T. brucei. Cloned stable transformants containing each of the eight tags were isolated. B. Construction of the plasmid piTag. Eight different 40mers were integrated into the plasmid upstream of the procyclin promoter. Expression of the neomycin resistance gene (NeoR) in trypanosomes is controlled by the EP1 procyclin promoter and 5′ untranslated region (UTR) and the last 19 bases of the 3′ untranslated region and intergenic region (IGR) of EP2 procyclin. The linearised plasmid integrates into an rDNA spacer in the genome. Tags were amplified from genomic DNA by nested PCR.