FIG. 3.

Stability of the mutation after long-term infection. PCR to discard that the in vivo persistence of the infection was due to the recovery of the wild-type phenotype by redundancy or selection of revertants. The reaction was performed using genomic DNA isolated from parasites recovered by hemoculture from chronically infected mice, with the L16 clone or the WT strain as the template. Four sets of primers were used: (i) 121F-122R, to check that the isolated DNA belonged to T. cruzi, (ii) N1N2 to amplify the neomycin gene (850 bp), (iii) H1H2 to amplify the hygromycin gene (960 bp), and (iv) LYT1LYT2 to amplify the complete CDS of LYT1 (1,659 bp). The presence of amplification products for the antibiotic resistance genes as well as the absence of amplification for the LYT1 gene on the L16 clone demonstrated that the genetic replacement kept stable. M, DNA size marker.