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. 2021 Apr 27;15(4):e0009377. doi: 10.1371/journal.pntd.0009377

Fig 3. Deletion of the DHFR-TS gene in Leishmania infantum.

Fig 3

(A) Schematic representation of the DHFR-TS locus and of the integration of the hygromycin phosphotransferase B (HYG) and neomycin phosphotransferase (NEO) deletion cassettes. Also shown are the expected sizes after digestion with XhoI and the location of probe 1 (small black boxes) used for the hybridization of Southern blots. (B) Southern blot analysis of genomic DNAs digested with XhoI from L. infantum wild-type (1), the single allele knock out DHFR-TSNEO/+ (2), or the aneuploid cell DHFR-TSNEO/HYG/+ (3) hybridized to probe 1. (C) Southern blot analysis of genomic DNAs digested with XhoI from L. infantum wild-type (1), the single allele knockout clone DHFR-TSNEO/+ transfected with psp72αblastα-DHFR-TS (4), or the DHFR-TSNEO/HYG null mutant harboring psp72αblastα-DHFR-TS (5) with probe 1. (D) Southern blot analysis of total DNAs digested with XbaI and HindIII (see S2A Fig) from a clone of the DHFR- TSNEO/HYG chromosomal null mutant harboring psp72αblastα-DHFR-TS (5) or of these cells grown for 20 (lane 6) and 25 (lane 7) passages without blasticidin but supplemented with 50 μg/ml of thymidine. This blot was hybridized to a DHFR-TS intragenic probe (see S2 Fig). The blot was re-hybridized with a PTR1 probe to monitor the amount of DNA analyzed (lower panel).