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. Author manuscript; available in PMC: 2012 May 1.
Published in final edited form as: J Microbiol Methods. 2011 Feb 23;85(2):103–113. doi: 10.1016/j.mimet.2011.02.001

Figure 6. Schematic representation of the PCR product mediated UPRT gene deletion in T. gondii.

Figure 6

A: Full-length disruption cassette amplified using DisFLF and DisFLR primers contains the T. gondii CAT selection cassette flanked at each side by ~ 2kb of genomic DNA from 5′ and 3′ region of UPRT gene. B: Strategy for split cassette mediated gene disruption in T. gondii. Combination of primers DisFLF and DisSplitR yielded UPRTDISCAS-A fragment which contains 5′ genomic region of the UPRT gene. Amplification using DisFLR and DisSpiltF using pUPRTDISMSG as the template resulted in the production of the UPRTDISCAS-B fragment. C: Three independent homologous recombination events are required for the reciprocal replacement of UPRT coding region in the genome with split cassette fragments: UPRIDSCAS-A and UPRTDISC-B. UPRT knock out parasites were enriched by growing the transfected parasites initially in the presence of chloramphenicol and then in the presence of 5mM 5-fluoro-2-deoxyuridine.