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. Author manuscript; available in PMC: 2014 Feb 1.
Published in final edited form as: Mol Microbiol. 2012 Dec 26;87(3):641–655. doi: 10.1111/mmi.12121

Figure 3. Construction of RH ΔTgAP2XI-4 using fusion PCR.

Figure 3

(A) In the first PCR, the 5′ upstream and 3′ downstream flanking regions of the TgAP2XI-4 gene were amplified along with the first and second half of a pyrimethamine resistant DHFR cassette. Due to the introduction of complementary flanking regions, the TgAP2XI-4 5′ and 3′ products could be fused to the first and second halves of the DHFR products, respectively, in a second PCR. The two resulting PCR products were directly transfected into the RH ΔKu80 T. gondii type I strain.

(B) PCR was used to confirm the correct integration of the fused ΔTgAP2XI-4 construct (using primer pairs ii and iii) and deletion of the TgAP2XI-4 coding sequence (primer pair i). The absence of TgAP2XI-4 transcripts was confirmed by RT-PCR (primer pair i). Primers for TgAP2XI-5 (TGME49_016220) were used as a control (con).