Somatic gene targeting with RNA/DNA chimeric oligonucleotides: an analysis with a sensitive reporter mouse system.
BACKGROUND
Targeted gene correction provides a potentially powerful method for gene therapy. RNA/DNA chimeric oligonucleotides were reported to be able to correct a point mutation with a high efficiency in cultured rodent cells, in the body of mice and rats, and in plants. The efficiency of correction in the liver of rats was claimed to be as high as 20% after tail-vein injection. However, several laboratories have failed to reproduce the high efficiency.


METHODS
In order to sensitively detect and measure sequence changes by the chimeric oligonucleotides, we used Muta Mouse, a transgenic mouse system for mutation detection in vivo. It carries, on its chromosome, multiple copies of the lambda phage genome with the lacZ(+) gene. Two chimeric oligonucleotides were designed to make a point mutation at the active site of the LacZ gene product. They were injected into the liver with HVJ liposomes, which were demonstrated to allow reliable gene delivery. One week later, DNA was extracted from the liver, and lambda::lacZ particles were recovered by in vitro packaging. The lacZ-negative phage was detected by selection with phenyl-beta-D-galactoside.


RESULTS
The mutant frequency of the injected mice was at the same level as the control mouse (approximately 1/10000). Our further restriction analysis and sequencing did not detect the designed mutations.


CONCLUSIONS
Gene correction frequency in mouse liver by these oligonucleotides was shown to be less than 1/20000 in our assay with the Muta Mouse system.
Ino
Asami
A
Department of Medical Genome Sciences, Graduate School of Frontier Science & Institute of Medical Science, University of Tokyo, 4-6-1, Shirokanedai, Minato-ku, Tokyo 108-8639, Japan.
Yamamoto
Seiji
S
Kaneda
Yasufumi
Y
Kobayashi
Ichizo
I
D000818:Animals; D010582:Bacteriophage lambda / Q000235:genetics; D001483:Base Sequence; D004260:DNA Repair; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D018390:Gene Targeting; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D007763:Lac Operon; D008081:Liposomes; D008099:Liver / Q000378:metabolism; D008297:Male; D051379:Mice; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D020319:Oligodeoxyribonucleotides, Antisense / Q000008:administration & dosage / Q000235:genetics; D016376:Oligonucleotides, Antisense / Q000008:administration & dosage / Q000235:genetics*; D020320:Oligoribonucleotides, Antisense / Q000008:administration & dosage / Q000235:genetics; D017354:Point Mutation
D005456:Fluorescent Dyes; D008081:Liposomes; D020319:Oligodeoxyribonucleotides, Antisense; D016376:Oligonucleotides, Antisense; D020320:Oligoribonucleotides, Antisense; D016650:Fluorescein-5-isothiocyanate