Each component of a 2A polycistronic expression cassette can be removed by using the appropriate restriction enzymes: (i) For Pdx1-E2A-eGFP, Pdx1-E2A can be cut out using SacI and NotI/BamHI (left), eGFP can be cut out using NotI/BamHI and SalI (middle) and Pdx1-E2A-eGFP can be cut out using SacI and SalI (ii) for Nkx2.2-T2A-eGFP, Nkx2.2-T2A can be cut out using NotI and BamHI (left), and eGFP can be cut out using BamHI and SalI (middle), and Nkx2.2-T2A-eGFP can be cut out using NotI and SalI (right) (iii) for Ngn3-F2A-eGFP, Ngn3-F2A can be cut out using BamHI and HindIII (left), eGFP can be cut out using HindIII and SalI (middle), and Ngn3-F2A-eGFP can be cut out using BamHI and SalI (right) (iv) for Nkx2.2-T2A-Ngn3-F2A-eGFP, Nkx2.2-T2A can be cut out using NotI and BamHI (left), Ngn3-F2A can be cut out using BamHI and HindIII (middle), and eGFP can be cut out using HindIII and SalI. ( C) Examples of the flexibility of the restriction enzyme digestion/ligation construction method. Each group of three lanes is (from left to right) molecular weight ladder, native sequence, and native sequence +2A. Note that the native Nkx2.2 amplified from MIN6 cell cDNA contained regions outside of the coding region of the mRNA, as the primers were optimised for maximum efficiency, and thus is larger in size compared to Nkx2.2-T2A. ( B) The amplification of Ngn3-F2A, Pdx1-E2A and Nkx2.2-T2A, a size difference between the native full-length gene and the gene with a 2A-peptide tag can be observed using gel electrophoresis. ( A) All 2A polycistronic expression cassettes were generated by restriction enzyme digestion and ligation utilising the restriction enzyme cutting sites: SacI, NotI, BamHI, HindIII and SalI (boxed, as described in Materials an methods) present in the multicloning site of the pCMV-Script plasmid (Stratagene). The construction of 2A polycistronic expression cassettes. ![]() The sequences of primers used in this study, the restriction enzyme sites used in the study are underlined.ĭifferent glycine-serine-glycine (GSG) linkers (Forward) and their corresponding reverse primer sequences (Reverse) used for the generation of 2A polycistronic constructs.
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