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. 2012 Sep 6;3(9):e385. doi: 10.1038/cddis.2012.113

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Copyright © 2012 Macmillan Publishers Limited

This work is licensed under the Creative Commons Attribution-NonCommercial-No Derivative Works 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-nd/3.0/

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Generation of MCA null mutants. (a) Schematic representation of the L. mexicana WT MCA locus and the constructs used for targeted gene replacement. ORFs are shown as arrows, 5′FR plus 5′ end of MCA, and 3′FR DNA sequences used for gene targeting are shown as boxes. Restriction sites within the WT locus and the construct are shown, and the predicted sizes after DNA digestion are indicated for both native and modified MCA locus. DHFR, dihydrofolate reductase gene. (b) Southern blot analysis. Genomic DNA was digested with AgeI/SacII and hybridised with a labelled DNA probe comprising the 5′region used for gene targeting (upper) or an ORF probe (lower). Molecular mass markers are shown on the left. WT (lane 1), heterozygote (lane 2) and Δmca (lane 3). (c and d) Western blot analysis. Whole-cell lysates of L. mexicana promastigotes were probed with anti-L. major MCA antibody. WT (lane 1), Δmca (lane 2), Δmca∷MCA (lane 3), WT (MCA) (lane 4) and WT (MCA^C201–202G) (lane 5). EF1-α was used as a loading control. (e) Growth curve of L. mexicana promastigotes: WT (▪), Δmca (▴), Δmca∷MCA (), WT (MCA) (), WT (MCA ^C201–202G)(•). (f) Growth curve of L. mexicana axenic amastigotes: WT (▪), Δmca (▴), Δmca∷MCA (), WT (MCA) (). ***, ** and * significant differences compared with WT (t-test, ***P<0.001, **P<0.01 and *P<0.05)

Inline graphic — Generation of MCA null mutants. (a) Schematic representation of the L. mexicana WT MCA locus and the constructs used for targeted gene replacement. ORFs are shown as arrows, 5′FR plus 5′ end of MCA, and 3′FR DNA sequences used for gene targeting are shown as boxes. Restriction sites within the WT locus and the construct are shown, and the predicted sizes after DNA digestion are indicated for both native and modified MCA locus. DHFR, dihydrofolate reductase gene. (b) Southern blot analysis. Genomic DNA was digested with AgeI/SacII and hybridised with a labelled DNA probe comprising the 5′region used for gene targeting (upper) or an ORF probe (lower). Molecular mass markers are shown on the left. WT (lane 1), heterozygote (lane 2) and Δmca (lane 3). (c and d) Western blot analysis. Whole-cell lysates of L. mexicana promastigotes were probed with anti-L. major MCA antibody. WT (lane 1), Δmca (lane 2), Δmca∷MCA (lane 3), WT (MCA) (lane 4) and WT (MCA^C201–202G) (lane 5). EF1-α was used as a loading control. (e) Growth curve of L. mexicana promastigotes: WT (▪), Δmca (▴), Δmca∷MCA (), WT (MCA) (), WT (MCA ^C201–202G)(•). (f) Growth curve of L. mexicana axenic amastigotes: WT (▪), Δmca (▴), Δmca∷MCA (), WT (MCA) (). ***, ** and * significant differences compared with WT (t-test, ***P<0.001, **P<0.01 and *P<0.05)