title: Designing and Cloning Molecular Constructs to Knock OutN-Acetylglucosamine Phosphatidylinositol De-N-Acetylase (GPI12) Gene inLeishmania major(MRHO/IR/75/ER).
abstract: BACKGROUND
  Leishmaniasis represents a major public health concern in tropical and sub-tropical countries. At present, there is no efficacious vaccine against the disease and new control methods are needed. One way to access this important goal is to knock out genes of specific macromolecules to evaluate the effect of deletion on the growth, multiplication, pathogenesis and immunity of the parasite. The aim of this study was to design and clone molecular constructs to knock outN-acetylglucosamine phosphatidylinositol de-N-acetylase(GPI12) gene inLeishmania major.
  
  
  METHODS
  For designing and making molecular constructs, we used pLEXSY-neo2 and pLEXSY-hyg2 vectors. The molecular constructs were cloned inE. colistrain Top10. The molecular constructs were transfected by electroporation intoL. majorin two stages.
  
  
  RESULTS
  The molecular constructs were confirmed by Colony PCR and sequencing. The recombinant strains were isolated by selective antibiotics, after which they were confirmed by PCR, Southern and Western blots.
  
  
  CONCLUSIONS
  Recombinant parasites were created and examined for subsequent study. With the use of molecular constructs, it was possible to remove and study geneGPI12and to achieve a live recombinantLeishmaniaparasite that maintained the original form of the antigenic parasites. This achievement can be used as an experimental model for vaccine development studies. Further investigations are essential to check this model in a suitable host.
authors.affiliation: Leishmaniasis Research Center, Kerman University of Medical Sciences, Kerman, Iran.
authors.forename: Pooya
authors.initials: P
authors.lastname: Ghasemi Nejad Almani
authors.affiliation: Leishmaniasis Research Center, Kerman University of Medical Sciences, Kerman, Iran.
authors.forename: Iraj
authors.initials: I
authors.lastname: Sharifi
authors.affiliation: Cellular and Molecular Biology Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran; Biotechnology Dept., School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
authors.forename: Bahram
authors.initials: B
authors.lastname: Kazemi
authors.affiliation: Leishmaniasis Research Center, Kerman University of Medical Sciences, Kerman, Iran.
authors.forename: Zahra
authors.initials: Z
authors.lastname: Babaei
authors.affiliation: Cellular and Molecular Biology Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran; Biotechnology Dept., School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
authors.forename: Mojgan
authors.initials: M
authors.lastname: Bandehpour
authors.affiliation: Research Center for Tropical and Infectious Diseases, Kerman University of Medical Sciences, Kerman, Iran.
authors.forename: Samira
authors.initials: S
authors.lastname: Salari
authors.affiliation: Leishmaniasis Research Center, Kerman University of Medical Sciences, Kerman, Iran.
authors.forename: Ebrahim
authors.initials: E
authors.lastname: Saedi Dezaki
keywords: Cloning; GPI12; Leishmaniasis; Molecular constructs