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. 2013 Apr;8(2):190-6.

Gene regulation of pteridine reductase 1 in leishmania promastigotes and amastigotes using a full-length antisense construct

F Kheirandish  1 , M Bandehpour, N Davoudi, N Mosaffa, S Dawood, B Kazemi, A Haghighi, A Khamesipour, H Masjedi, M Mohebali, F Mahboudi
Affiliations

Gene regulation of pteridine reductase 1 in leishmania promastigotes and amastigotes using a full-length antisense construct

F Kheirandish et al. Iran J Parasitol. 2013 Apr.

Abstract

Background: Pteridine metabolic pathway is unusual features of Leishmania, which is necessary for the growth of parasite. Leishmania has evolved a complex and versatile pteridine salvage network which has the ability of scavenging a wide area of the conjugated and unconjugated pteridines especially folate and biopterin. In this study, we focus on the inhibition of ptr1 gene expression.

Methods: L. major ptr1 gene was cloned into pcDNA3 and digested using KpnI and BamHI. The gene was subcloned so that antisense will transcribe and called pcDNA-rPTR. Leishmania major was cultured and late logarithmic-phase promastigotes were harvested. The promastigotes were divided into two groups. One group was transfected with 50 µg of pcDNA-rPTR, whereas the other group was transfected with pcDNA3. Transfected cells were cultured and plated onto semi-solid media. Mouse pritonean macrophages were transfected using pcDNA-rPTR-tansfected promastigotes. Western blotting was performed on mouse transfected pritonean macrophages and extracts from transfected promastigotes of L. major using a L. major ptr1 antibody raised in rabbits.

Results: The PTR1 protein was not expressed in pcDNA-rPTR- tansfected promastigotes and mouse macrophage transfected with pcDNA-rPTR- tansfected promastigotes.

Conclusion: This approach might be used to study the pteridine salvage pathway in Leishmania or to assess the possibility of using gene expression inhibition in the treatment of leishmaniasis.

Keywords: Antisense; Gene regulation; Inhibition; Leishmania; Pteridine reductase 1.

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Figures

Fig. 1
Fig. 1
Electrophoresis of digested pBSC-ptr using enzyme/ Lane 1: Digested pBSC-ptr by KpnI and BamHI/Lane 2: 100-bp DNA ladder marker
Fig. 2
Fig. 2
Identification of recombinant plasmid pcDNA-rPTR using enzyme digestion. Lane 1:100-bp DNA ladder marker. Lane 2: pcDNA-rPTR digested with KpnI and BamHI
Fig. 3
Fig. 3
Western blot analysis to demonstrate lack of expression of PTR1 protein in antisense-transfected promastigotes Lane 1: Lysate of pcDNA3-transfected promastigotes Lane 2: Lysate of pcDNA-rPTR-transfected promastigotes
Fig. 4
Fig. 4
Analysis of expression protein by Western blotting Lane 1: Infected macrophages by amastigotes containing pcDNA3 /Lane 2: Extract macrophages Lane 3: Infected macrophages by amastigotes containing antisense (pcDNA-rPTR)
See this image and copyright information in PMC

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