Format

Send to

Choose Destination
See comment in PubMed Commons below
Neurosci Res. 2014 Mar;80:91-4. doi: 10.1016/j.neures.2013.11.007. Epub 2013 Dec 27.

A simple and highly efficient method to identify the integration site of a transgene in the animal genome.

Author information

1
Laboratory of Molecular Neuroimaging, Graduate School of Medical and Dental Sciences, Niigata University, Niigata, Niigata 951-8510, Japan.
2
Department of Comparative and Experimental Medicine, Brain Research Institute, Niigata University, Niigata 951-8510, Japan.
3
Division of Molecular and Cellular Biology, Graduate School of Medical and Dental Sciences, Niigata University, Niigata, Niigata 951-8510, Japan.
4
Laboratory of Molecular Neuroimaging, Graduate School of Medical and Dental Sciences, Niigata University, Niigata, Niigata 951-8510, Japan. Electronic address: masashi.kishi@gmail.com.

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.

KEYWORDS:

Insertion effect; Inverse PCR; NSE-tTA; Transgenic mouse; tetO-Cre

PMID:
24378375
DOI:
10.1016/j.neures.2013.11.007
[Indexed for MEDLINE]
PubMed Commons home

PubMed Commons

0 comments
How to join PubMed Commons

    Supplemental Content

    Full text links

    Icon for Elsevier Science
    Loading ...
    Support Center