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. 2016 Jan 19;6:1560. doi: 10.3389/fmicb.2015.01560

Figure 3.

Figure 3

Generation of a TgMCA mutant strain. (A,B) Schematic of the experimental design of the TgMCA knockout strain. A knockout vector (PTCR-CD TgMCA KO) was constructed to target the TgMCA complete gene. (C) Genomic PCR analysis of ΔTgMCA strain. The position of the primers are shown in (B). P1, P2 and P3, P4 were used to amplify exon2 to exon3 and exon10 to exon11 of TgMCA, respectively. (D) Western blot performed with anti-rTgMCA antibody on total extracts from Δku80 and ΔTgMCA, with TgActin used as control. (E) Schematic of the complementary strain construction. Crispr/Cas9-UPRT was used to target the UPRT locus of ΔTgMCA, and TgMCA coding sequence fused with the HA-tag at the C-terminal under the GRA1 promoter was inserted into the gap of the UPRT locus. (F) Western blot analysis of the restoration of TgMCA with anti-rTgMCA serum and anti-HA antibody on total lysate of Δku80 and ΔTgMCA-cm, with TgActin used as control.