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. 2008 May 13;69(1):152–163. doi: 10.1111/j.1365-2958.2008.06271.x

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© 2008 The Authors Journal compilation © 2008 Blackwell Publishing Ltd

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Fig. 3 — Targeted deletion of PySAP1.

A. Schematic representation of the replacement strategy to generate Pysap1(−) parasites. The endogenous PySAP1 genomic locus is targeted with a replacement fragment containing the 5′ and 3′ sequence within exon 1 of PySAP1 flanking the Toxoplasma gondii DHFR/TS-positive selection marker. Diagnostic wild type-specific or integration-specific test amplicons are indicated by lines.

B. PCR genotyping shows the gene replacement using oligonucleotide primer combinations that can only amplify from the recombinant locus (Test 1 and Test 2). The wild type-specific PCR reaction (WT) confirms the absence of wild-type parasites in the clonal Pysap1(−) parasites.

C. RT-PCR analysis (35 cycles) shows the loss of PySAP1 transcripts in RNA isolated from Pysap1(−) sgSPZs. The PySAP1-specific amplicon used for the analysis is shown above in (A) as the wild type (WT) test. PyCSP was used as a positive control.