کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
5915690 | 1163321 | 2011 | 4 صفحه PDF | دانلود رایگان |
Targeted gene replacement is a powerful tool in Leishmania genetics that can be time-consuming to implement. One tedious aspect that delays progress is the multi-step construction of gene targeting vectors. To accelerate this process, we developed a streamlined method that allows the assembly of a complete targeting vector from all its constituent parts in a single-step multi-fragment ligation. The individual components to be assembled are flanked by sites for the restriction endonuclease SfiI that generates non-identical, non-palindromic three base 3â²-overhangs designed to allow annealing and ligation of the parts only in the proper order. The method was optimized by generating constructs for targeting the Leishmania donovani inosine monophosphate dehydrogenase gene (LdIMPDH) encoding six different drug resistance markers, and was found to be rapid and efficient. These constructs were successfully employed to generate heterozygous LdIMPDH gene replacement mutants. This method is adaptable for generating targeting vectors for a variety of species.
36Research highlightsⶠA novel method is presented for rapidly constructing gene targeting vectors. ⶠThe method relies on SfiI to generate unique non-palindromic overhangs. ⶠThe gene targeting vectors are assembled in a single-step, multi-fragment ligation.
Journal: Molecular and Biochemical Parasitology - Volume 175, Issue 2, February 2011, Pages 209-212