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. 2019 Apr 26;9:126. doi: 10.3389/fcimb.2019.00126

Figure 5.

Figure 5

Generation of miltefosine transporter disrupted L. passim by CRISPR/Cas9-induced homology directed repair. (A) Schematic representation of the strategy to truncate L. passim miltefosine transporter (LpMT) by CRISPR/Cas9-induced homology directed repair (HDR). The positions of primers to detect wild type (WT) and truncated (Truncation) alleles of LpMT are shown with the expected sizes of PCR products. Five prime and 3′ untranslated regions (UTR) as well as open reading frame (ORF) of LpMT are shown in blue, green, and yellow, respectively. Donor DNA contains a hygromycin resistance gene (Hph, red) flanked by the parts of 5′ UTR and ORF of LpMT. The putative cleavage site by Cas9 is at 1,147 bp from the start codon of LpMT. Eleven drug (blasticidin, G418, and hygromycin) resistant clones (1–11) together with wild type L. passim were analyzed by genomic PCR to detect WT (851 bp amplicon) and Truncation (1,230 bp amplicon) alleles of LpMT. The positions of 500, 1,000, and 3,000 bp bands in molecular weight marker (MW) are shown at the left. (B) Detection of LpMT and GAPDH mRNAs in LpMT heterozygous (+/–) and homozygous (–/–) null mutants together with wild type L. passim (+/+) by RT-PCR. The expected sizes of RT-PCR products for LpMT and GAPDH are 564 and 279 bp, respectively. The negative control was run using water as the template (-RT) for RT-PCR. The positions of 200–700 bp bands in molecular weight marker (MW) are shown at the right.