title: Site-specific chromosomal integration in mammalian cells: highly efficient CRE recombinase-mediated cassette exchange.
abstract: Expression of experimental constructs in mammalian cells or transgenic animals is difficult to control because it is markedly influenced by position effects. This has limited both the analysis of cis -DNA regulatory elements for transcription and replication, and the physiological analysis of proteins expressed from transgenes. We report here two new methods based on the concept of recombinase-mediated cassette exchange (RMCE) to perform site-specific chromosomal integration. The first method permits the exchange of a negative selectable marker pre-localized on the chromosome with a transgene via a CRE-mediated double recombination between inverted Lox sites. Integration efficiency is close to 100 % of negatively selected mouse erythroleukemia cells and ranges from 10 to 50 % in embryonic stem cells. The second method allows RMCE with no selection at all except for cells that have taken up plasmid transiently. While less efficient, this technique permits novel experimental approaches. We find that integration of a transgene at a given genomic site leads to reproducible expression. RMCE should be useful to develop artificial genetic loci that impart specific and reproducible regulation of transgenes in higher eukaryotes. This should facilitate the analysis of cis -regulatory DNA elements governing expression and position effects, improve our control over the physiological effects of transgenes, and accelerate the development of animal models for complex human diseases.
authors.affiliation: Department of Medicine/Division of Hematology, Albert Einstein College of Medicine, Bronx, USA.
authors.forename: Y Q
authors.initials: YQ
authors.lastname: Feng
authors.forename: J
authors.initials: J
authors.lastname: Seibler
authors.forename: R
authors.initials: R
authors.lastname: Alami
authors.forename: A
authors.initials: A
authors.lastname: Eisen
authors.forename: K A
authors.initials: KA
authors.lastname: Westerman
authors.forename: P
authors.initials: P
authors.lastname: Leboulch
authors.forename: S
authors.initials: S
authors.lastname: Fiering
authors.forename: E E
authors.initials: EE
authors.lastname: Bouhassira
mesh_terms: D000818:Animals; D001287:Attachment Sites, Microbiological / Q000235:genetics; D002875:Chromosomes / Q000235:genetics*; D002999:Clone Cells / Q000378:metabolism; D003001:Cloning, Molecular; D015870:Gene Expression; D017930:Genes, Reporter / Q000235:genetics; D005819:Genetic Markers / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics; D016678:Genome; D019426:Integrases / Q000378:metabolism*; D051379:Mice; D016254:Mutagenesis, Insertional / Q000235:genetics / Q000379:methods*; D016297:Mutagenesis, Site-Directed / Q000235:genetics*; D011995:Recombination, Genetic / Q000235:genetics; D013234:Stem Cells / Q000378:metabolism; D014162:Transfection; D019076:Transgenes / Q000235:genetics; D014407:Tumor Cells, Cultured; D014764:Viral Proteins
chemical_list: D005819:Genetic Markers; D014764:Viral Proteins; C045073:Cre recombinase; D019426:Integrases