Research

A trial of somatic gene targeting in vivo with an adenovirus vector

Asami Ino^1,2 , Yasuhiro Naito^1,3 , Hiroyuki Mizuguchi⁴ , Naofumi Handa¹ , Takao Hayakawa⁵ and Ichizo Kobayashi^1,2

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

author email corresponding author email

Genetic Vaccines and Therapy 2005, 3:8doi:10.1186/1479-0556-3-8

Published:	12 October 2005

Abstract

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.