CIRCULAR EXPRESSION CONSTRUCTION FOR APPLICATIONS OF GENE THERAPY.

摘要

Procedure for the preparation of a circular expression construct, closed in the form of a ring, from a double strand of DNA, consisting of the following steps: a) Cutting a double stranded DNA sequence from an amplifiable plasmid in prokaryotic cells or eukaryotic by a primary digestion with restriction endonucleases, b) in which the cut sites directly limit on both sides and without intercalated bases the sequences of an expression cassette composed of i. at least one promoter sequence, ii. at least one coding sequence and iii. at least one polyadenylation sequence, and c) following intramolecular ligation of the restriction fragments generated such that the ligation reaction provides a double strand of covalently closed DNA (ring closure), followed by d) secondary digestion of the mixture of reaction with a restriction endonuclease that recognizes and cuts a recognition sequence that is not present in the expression construct to be prepared but does appear at least once in the rest of the biologically amplifiable plasmid, and e) simultaneous or subsequent degradation of the biologically multiplied plasmid open chain moiety with a specific exonuclease for the 3 '' '' and 5 '' '' ends of the DNA and f) purification of the closed, ring-shaped expression cassette of a double strand of DNA.