Iranian
Biomedical Journal
Volume 7, Issue 1 (1-2003)
IBJ 2003, 7(1): 13-18 |
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Davoudi N, Mahboudi F, Azizi M, Adeli A, McMaster R W. Introduction of Three Independent Selection Markers in Leishmania. IBJ 2003; 7 (1) :13-18
URL: http://ibj.pasteur.ac.ir/article-1-547-en.html
URL: http://ibj.pasteur.ac.ir/article-1-547-en.html
Abstract:
The pLE2SCX vector was developed for the stable expression of exogenous genes in the protozoan parasite Leishmania. The pLE2SCX construct contains three independent selection markers: herpes simplex virus thymidine kinase (HSV-TK), cytosine deaminase (CD) and streptothericin acetyltransferase gene (sat) in multiple cloning site, flanking by 5’ and 3’ untranslated regions of the previously cloned Leishmania major hexa-binding protein gene. Selection was based on resistance to the nourseothericin (Ns) which corresponds to sat gene. The two negative selection HSV-TK and CD genes, make the transformed cell sensitive to ganciclovir (GCV) and 5-fluorocytosine (5-FC). The vector was introduced into Leishmania promastigotes by electroporation and maintained as circular form. The selected transfectants were not grown on media with GCV or 5-FC. Using two drug sensitive selectable markers together on a vector is a novel strategy in gene cloning in Leishmania. This stable transfection vector has allowed the permanently expression of several different exogenous genes at the same time in Leishmania
Keywords: HSV-thymidine kinase (HSV-TK), Cytosine deaminase (CD), ganciclovir (GCV), 5-fluorocytosine (5-FC), nourseothericin (Ns)
Type of Study: Full Length/Original Article |
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