Tehran University of Medical Sciences Iranian Journal of Parasitology 1735-7020 11 4 2016 12 25 448 462 Pooya GHASEMI NEJAD ALMANI Leishmaniasis Research Center, Kerman University of Medical Sciences, Kerman, Iran Iraj SHARIFI Leishmaniasis Research Center, Kerman University of Medical Sciences, Kerman, Iran Bahram KAZEMI Cellular and Molecular Biology Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran AND Biotechnology Dept., School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran Zahra BABAEI Leishmaniasis Research Center, Kerman University of Medical Sciences, Kerman, Iran Mojgan BANDEHPOUR Cellular and Molecular Biology Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran AND Biotechnology Dept., School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran Samira SALARI Research Center for Tropical and Infectious Diseases, Kerman University of Medical Sciences, Kerman, Iran Ebrahim SAEDI DEZAKI Leishmaniasis Research Center, Kerman University of Medical Sciences, Kerman, Iran 2016 12 25 2016 12 25 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 out N-acetylglucosamine phosphatidylinositol de-N-acetylase (GPI12) gene in Leishmania major. Methods: For designing and making molecular constructs, we used pLEXSY-neo2 and pLEXSY-hyg2 vectors. The molecular constructs were cloned in E. coli strain Top10. The molecular constructs were transfected by electroporation into L. major in 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. Conclusion: Recombinant parasites were created and examined for subsequent study. With the use of molecular constructs, it was possible to remove and study gene GPI12 and to achieve a live recombinant Leishmania parasite 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. https://ijpa.tums.ac.ir/index.php/ijpa/article/view/1344 https://ijpa.tums.ac.ir/index.php/ijpa/article/download/1344/622