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Centre National De La Recherche Scientifique

Institut Pasteur

Institut Curie

Frederic Jaisser

Andre Choulika

Sylvie Robine

Daniel Louvard

Charles Babinet

Michel Cohen-Tannoudji

The invention relates to methods of introducing a heterologous DNA sequence into a mouse embryonic stem cell wherein the DNA sequence is inserted by homologous recombination into a villin gene/I-SceI hybrid by creating a double strand break with I-SceI meganuclease. Subsequently, the mouse embryonic stem cells can be used to generate a transgenic mouse comprising the heterologous DNA sequence. Additionally, the methods can be used for gene replacement in ovo where a mouse oocyte containing a villin gene/I-SceI hybrid within its genome exists or is first generated. More generally, the methods can be used for the targeted insertion of a heterologous DNA sequence into any cell containing a villin gene/I-SceI hybrid sequence within its genome.

I-Sce I induced gene replacement and gene conversion in embryonic stem cells

This invention relates to a method of constructing a villin gene hybrid by inserting an I-Sce I restriction site next to or within a gene or cDNA encoding a villin protein. The insertion site of the I-Sce I restriction site is chosen as to provide a first downstream part and a second upstream part from the site, containing at least twelve nucleotides of the gene or cDNA encoding the villin protein. Furthermore, the insertion of the restriction permits a high frequency of homologous recombination events. The villin gene hybrid may be used to transfect eukaryotic cells, and particularly, embryonic stem cells.

I Sce I-induced gene replacement and gene conversion in embryonic stem

Growing community of inventors

Malakoff, France

Frederic Jaisser