A new method (GOREC) for directed mutagenesis and gene repair by homologous recombination.
Directed mutagenesis in mammalian cells has been the focus of intense research because of its promising application for gene correction and engineering. Both natural and modified oligonucleotides (ODN), RNA-DNA chimeric oligonucleotide (RDO) and small fragment DNA (SFHR), as well as vector DNA were used for promoting homologous replacement with varying success. It was recently shown that a triple helix-forming oligonucleotide (TFO) tethered to an oligonucleotide (donor DNA) can enhance mutagenesis by homologous recombination in cells. The basic idea is to accelerate homology search by oligonucleotide-directed triple helix formation in the vicinity of the target site for donor DNA. Here we describe a new method named GOREC (guided homologous recombination) which shares similar gene targeting, but has notable difference in the concept with the previous method. It is made of a homing device (TFO) and a donor DNA for effecting distinct functions. They are linked together by non-covalent or covalent interaction. This modular concept allows guidance of either an oligonucleotide (ODN, RDO) or a small DNA fragment to the target site for homologous replacement. Therefore, the triple helix site can be hundreds of base pairs away from the target site. An episomal assay for proof-of-principle study will be presented and discussed.
Maurisse
R
R
Laboratoire de Biophysique, UMR8646 CNRS-Muséum National d'Histoire Naturelle, U201 INSERM, Paris, France.
Feugeas
J-P
JP
Biet
E
E
Kuzniak
I
I
Leboulch
P
P
Dutreix
M
M
Sun
J-S
JS
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D004247:DNA / Q000008:administration & dosage; D018390:Gene Targeting / Q000379:methods*; D015316:Genetic Therapy / Q000379:methods*; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D009841:Oligonucleotides / Q000235:genetics; D011995:Recombination, Genetic; D014184:Transplantation, Homologous
D008164:Luminescent Proteins; D009841:Oligonucleotides; D049452:Green Fluorescent Proteins; D004247:DNA