Efficient in toto targeted recombination in mouse liver by meganuclease-induced double-strand break.
BACKGROUND
Sequence-specific endonucleases with large recognition sites can cleave DNA in living cells, and, as a consequence, stimulate homologous recombination (HR) up to 10 000-fold. The recent development of artificial meganucleases with chosen specificities has provided the potential to target any chromosomal locus. Thus, they may represent a universal genome engineering tool and seem to be very promising for acute gene therapy. However, in toto applications depend on the ability to target somatic tissues as well as the proficiency of somatic cells to perform double-strand break (DSB)-induced HR.


METHODS
In order to investigate DSB-induced HR in toto, we have designed transgenic mouse lines carrying a LagoZ gene interrupted by one I-SceI cleavage site surrounded by two direct repeats. The LagoZ gene can be rescued upon cleavage by I-SceI and HR between the two repeats in a process called single-strand annealing. beta-Galactosidase activity is monitored in liver after tail vein injection of adenovirus expressing the meganuclease I-SceI.


RESULTS
In toto staining revealed a strong dotted pattern in all animals injected with adenovirus expressing I-SceI. In contrast, no staining could be detected in the control. beta-Galactosidase activity in liver extract, tissue section staining, and PCR analysis confirmed the presence of the recombined LagoZ gene.


CONCLUSIONS
We demonstrate for the first time that meganucleases can be successfully delivered in animal and induce targeted genomic recombination in mice liver in toto. These results are an essential step towards the use of designed meganucleases and show the high potential of this technology in the field of gene therapy.
Gouble
Agnès
A
CELLECTIS S.A., 102 route de Noisy, 93235 Romainville, France.
Smith
Julianne
J
Bruneau
Sylvia
S
Perez
Christophe
C
Guyot
Valérie
V
Cabaniols
Jean-Pierre
JP
Leduc
Sophie
S
Fiette
Laurence
L
Avé
Patrick
P
Micheau
Béatrice
B
Duchateau
Philippe
P
Pâques
Frédéric
F
D000818:Animals; D001483:Base Sequence; D004247:DNA / Q000235:genetics / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D017930:Genes, Reporter; D005818:Genetic Engineering; D015316:Genetic Therapy; D007763:Lac Operon; D008099:Liver / Q000378:metabolism; D051379:Mice; D008822:Mice, Transgenic; D011995:Recombination, Genetic; D029701:Saccharomyces cerevisiae Proteins
D029701:Saccharomyces cerevisiae Proteins; D004247:DNA; C037725:SCEI protein, S cerevisiae; D015252:Deoxyribonucleases, Type II Site-Specific