Generation of <i>TcAPx</i> null mutants.

Abstract

<p><b>A</b> Map of the <i>T</i>. <i>cruzi</i> Sylvio X10/6 <i>TcAPx</i> locus (1) indicating the derivation of constructs used for targeted integration. The location of the flanking <i>TcCLPTM1</i> (Cleft-lip and palate transmembrane 1–like protein) and <i>TcG6PDH</i> (glucose-6-phosphate dehydrogenase) genes are indicated and the hatched box represents a degenerate VIPER/SIRE element. The ‘first round’ gene disruption construct is shown in (2), with the ‘second round’ gene deletion vector represented by (3). Restriction sites shown are C: Cla I and S: Sma I. <b>B</b> Strategy used to generate TcAPx null mutants. Briefly, the first allele was disrupted by insertional integration of the <i>PAC</i> gene into the ORF (1). An episomal copy of TcAPx was introduced into the <i>TcAPX</i>^+/- heterozygote line (2). The second endogenous allele was then deleted by homologous recombination using the flanking DNA external to the ORF to insert the <i>BLA</i> gene (3). The parasites were then removed from G418 selective pressure and passaged for up to 125 generations (4). Clones were then isolated and characterised (5). <b>C</b> The pTEX-APx episome is unstable in both wild type and <i>TcAPx</i> null backgrounds. The autoradiographs show Southern blots containing genomic DNA from wild type and null mutant cells isolated before (lanes 0) and after removal of G418 from the growth medium. Generations without G418 selection are indicated above the blot. The blot was probed with the <i>Neo</i>^R ORF. <b>D</b> Western blot showing expression level of TcAPx in parasite populations 125 generations after removal of G418 selection. WT: wild type Sylvio X10/6 lysate; SK: lysate from cells with a single copy of <i>TcAPX</i> disrupted; TcΔAPx: lysate from cells with both genes ablated. The panel below shows the blot probed with anti-TbBiP (a kind gift from Jay Bangs, University of Wisconsin-Madison) to control for loading. <b>E</b> Southern blot of genomic DNA digested with Cla I and Sma I, showing that <i>TcAPx</i> is absent from cloned null mutant cells. The left hand panel was probed with the <i>TcAPx</i> ORF and shows the endogenous gene (lane WT, 2.8 kb Cla I-Sma I fragment) and the gene disrupted by <i>PAC</i> insertion (lanes TcΔAPx 1 and 2, 3.1 kb Cla I fragment). <b>F</b> Western blot indicating that the null mutant clones do not express TcAPx. The wild type population show a single band of ~30kDa (lane WT: wild type) which is absent from the null mutant clones (lanes TcΔAPx 1 and 2). Equivalent loading is indicated by the Coomassie stained gel below.</p