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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.

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