Open AccessElimination of the plasmid bacterial backbone in site-directed transgenesis
Author(s) -
Jannik E. Jakobsen,
Jacob Giehm Mikkelsen,
Anders Lade Nielsen
Publication year - 2010
Publication title -
biotechniques/biotechniques
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.617
H-Index - 131
eISSN - 1940-9818
pISSN - 0736-6205
DOI - 10.2144/000113386
Subject(s) - transgene , plasmid , transgenesis , minicircle , biology , expression cassette , recombinase , site specific recombination , recombineering , dna , genetics , cre recombinase , gene , computational biology , microbiology and biotechnology , recombinant dna , recombination , genetically modified mouse , vector (molecular biology) , embryogenesis , reproductive biology
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.