Elimination of the plasmid bacterial backbone in site-directed transgenesis.
For cellular and animal transgenesis, FLP- and Cre-recombinase gene capture systems are highly effective to provide stable integration of a donor plasmid carrying the transgene cassette of interest into an engineered genomic locus in a given cell line. However, in many protocols, the entire plasmid bacterial backbone is integrated along with the transgene cassette. Here, we present a very simple yet highly efficient method for excluding plasmid bacterial backbone integration. The transgene cassette, including a single FLP recognition target site, is specifically amplified by PCR, and the resulting DNA ligated into minicircles can serve as donor DNA in FLP-mediated recombination. Interestingly, the elimination of the bacterial backbone increased expression of the inserted transgene. The presented method is simple and efficient for generating transgene cassette insertions devoid of the bacterial backbone.
Jakobsen
Jannik
J
Department of Human Genetics, University of Aarhus, Denmark.
Mikkelsen
Jacob
J
Nielsen
Anders
A
D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods*; D004254:DNA Nucleotidyltransferases / Q000235:genetics; D004262:DNA Restriction Enzymes / Q000378:metabolism; D004270:DNA, Circular / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D018014:Gene Transfer Techniques; D017930:Genes, Reporter / Q000235:genetics; D005822:Genetic Vectors; D006801:Humans; D008957:Models, Genetic; D010957:Plasmids / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016133:Polymerase Chain Reaction / Q000379:methods
D004270:DNA, Circular; D004254:DNA Nucleotidyltransferases; C045119:FLP recombinase; D004262:DNA Restriction Enzymes