Research
A trial of somatic gene targeting in vivo with an adenovirus vector
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* Corresponding author: Ichizo Kobayashi ikobaya@ims.u-tokyo.ac.jp
1 Department of Medical Genome Sciences, Graduate School of Frontier Science, University of Tokyo & Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan
2 Graduate Program in Biophysics and Biochemistry, Graduate School of Science the University of Tokyo
3 Department of Environmental Information, Keio University, 5322 Endo, Fujisawa, Kanagawa 252-8520, Japan
4 Laboratory of Gene Transfer and Regulation, National Institute of Biomedical Innovation, Asagi 7-6-8, Saito, Ibaraki, Osaka 567-0085, Japan
5 Pharmaceuticals and Medical Devices Agency, Shin-Kasumigaseki Bldg. 3-3-2, Kasumigaseki, Chiyoda-ku, Tokyo 100-0013, Japan
Genetic Vaccines and Therapy 2005, 3:8 doi:10.1186/1479-0556-3-8
Published: 12 October 2005Abstract
Background
Gene targeting in vivo provides a potentially powerful method for gene analysis and gene therapy. In order to sensitively detect and accurately measure designed sequence changes, we have used a transgenic mouse system, MutaMouse, which has been developed for detection of mutation in vivo. It carries bacteriophage lambda genome with lacZ+ gene, whose change to lacZ-negative allele is detected after in vitro packaging into bacteriophage particles. We have also demonstrated that gene transfer with a replication-defective adenovirus vector can achieve efficient and accurate gene targeting in vitro.
Methods
An 8 kb long DNA corresponding to the bacteriophage lambda transgene with one of two lacZ-negative single-base-pair-substitution mutant allele was inserted into a replication-defective adenovirus vector. This recombinant adenovirus was injected to the transgenic mice via tail-vein. Twenty-four hours later, genomic DNA was extracted from the liver tissue and the lambda::lacZ were recovered by in vitro packaging. The lacZ-negative phage was detected as a plaque former on agar with phenyl-beta-D-galactoside.
Results
The mutant frequency of the lacZ-negative recombinant adenovirus injected mice was at the same level with the control mouse (~1/10000). Our further restriction analysis did not detect any designed recombinant.
Conclusion
The frequency of gene targeting in the mouse liver by these recombinant adenoviruses was shown to be less than 1/20000 in our assay. However, these results will aid the development of a sensitive, reliable and PCR-independent assay for gene targeting in vivo mediated by virus vectors and other means.