Identification and Functional Analysis of Trypanosoma cruzi Genes That Encode Proteins of the Glycosylphosphatidylinositol Biosynthetic Pathway
Figure 5
Generation of TcGPI8 heterozygous mutants.
(A) DNA constructs generated to delete both TcGPI8 alleles by homologous recombination are shown with the NeoR or HygR genes flanked by 5′ and 3′ sequences of the TcGPI8 gene and the SacI/SpeI and XhoI/XbaI cloning sites from the pCR2.1TOPO vector. After transfecting epimastigotes with the purified DNA fragments, parasites were selected in LIT medium containing 200 µg/ml of G418 or hygromycin. Total DNA, isolated from G418 or hygromycin resistant parasites was analyzed by PCR amplifications, using the primers indicated by arrows. Below the schemes of DNA constructs, the sizes of the NeoR or HygR genes and the 5′ and 3′ sequences of the TcGPI8 gene are shown. (B) PCR amplification products analyzed on 1% agarose gel electrophoresis were obtained from DNA isolated from epimastigotes transfected with the GPI8-Neo (top panel) or GPI8-Hyg construct (bottom panel) and using pairs of primers showed in A. Amplicons derived from PCR using the primer pair 1F/7R that amplify a T. cruzi GPI8 allele which was not deleted are shown. On lanes indicated by (-), loaded samples were from PCR in which no template DNA was added. (C) Expression levels of TcGPI8 mRNA in WT and TcGPI8 single knockout of each allele interrupted by NeoR or HygR genes (+/− N or +/− H, respectively). Total RNAs purified from epimastigotes were hybridized to [α-32P]-labeled probes specific for the TcGPI8 gene (top panel) or for the 24Sα rRNA (bottom panel) used as loading control. The size of ribosomal RNA bands are indicated on the left and a graph with the quantification of the signals from the TcGPI8 probe after normalization using the 24Sα rRNA probe is shown below.