title: A simple and highly efficient method to identify the integration site of a transgene in the animal genome.
abstract: Because genetic manipulation occasionally disrupts the expression of the neighboring genes, the chromosomal locus where the transgene has been integrated should be identified in the use of transgenic organisms. By using a new blend of thermostable DNA polymerase, we established a highly efficient method of inverse polymerase chain reaction for this purpose. By using this protocol, we successfully determined the vector integration sites of 2 mouse lines, NSE-tTA and tetO-Cre, the combination of which is a useful tool in neuroscience research. On the basis of this information, we quantified the relative expression amount of the chromosomal genes adjacent to these transgenes and found that the insertion of the tetO-Cre vector significantly altered the mRNA level of one of the examined genes. Considering the potential risk of the insertion effect, we recommend that the vector integration sites of any transgenic lines should be determined routinely by using this method, and that the expression levels of their neighboring genes should be determined.
authors.affiliation: Laboratory of Molecular Neuroimaging, Graduate School of Medical and Dental Sciences, Niigata University, Niigata, Niigata 951-8510, Japan.
authors.forename: Shun
authors.initials: S
authors.lastname: Uemura
authors.affiliation: Laboratory of Molecular Neuroimaging, Graduate School of Medical and Dental Sciences, Niigata University, Niigata, Niigata 951-8510, Japan.
authors.forename: Tadahiro
authors.initials: T
authors.lastname: Nagaoka
authors.affiliation: Department of Comparative and Experimental Medicine, Brain Research Institute, Niigata University, Niigata 951-8510, Japan.
authors.forename: Minesuke
authors.initials: M
authors.lastname: Yokoyama
authors.affiliation: Division of Molecular and Cellular Biology, Graduate School of Medical and Dental Sciences, Niigata University, Niigata, Niigata 951-8510, Japan.
authors.forename: Michihiro
authors.initials: M
authors.lastname: Igarashi
authors.affiliation: Laboratory of Molecular Neuroimaging, Graduate School of Medical and Dental Sciences, Niigata University, Niigata, Niigata 951-8510, Japan. Electronic address: masashi.kishi@gmail.com.
authors.forename: Masashi
authors.initials: M
authors.lastname: Kishi
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D018014:Gene Transfer Techniques; D017930:Genes, Reporter / Q000235:genetics*; D019426:Integrases / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008822:Mice, Transgenic; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D019076:Transgenes / Q000235:genetics*
chemical_list: D001426:Bacterial Proteins; D002352:Carrier Proteins; C083139:Tet O resistance protein, Bacteria; D014157:Transcription Factors; C045073:Cre recombinase; D019426:Integrases
keywords: Insertion effect; Inverse PCR; NSE-tTA; Transgenic mouse; tetO-Cre