Leishmania major HEXBP deletion mutants generated by double targeted gene replacement

Abstract

The Leishmania major single-stranded DNA binding protein HEXBP contains nine 'CCHC' zinc finger motifs and binds to oligodeoxynucleotides derived from the antisense strand of the GP63 gene 5' flanking region in gel mobility shift assays and UV-crosslinking assays. In the present study a HEXBP-deficient clone of L. major was generated by double targeted gene replacement. The two HEXBP alleles were sequentially replaced with genes encoding resistance to the aminoglycoside antibiotics hygromycin B and G418 and drug-resistant clones were selected by plating on semi-solid drug-containing media. Successful deletion of both copies of the HEXBP gene implies that HEXBP is a not essential for growth of Leishmania promastigotes. Characterization HEXBP-deficient promastigotes revealed that HEXBP deficiency had no effect on the abundance of GP63 mRNA and protein in in vitro cultivated promastigotes and that HEXBP-deficient promastigotes were capable of lesion formation in BALB/c mice.