A tool for enhancing site-specific gene integration in mammalian cells.

Abstract

Efficient gene targeting, i.e., recombination of a chromosomal locus with a transfected DNA molecule, would allow deleterious mutations to be corrected in situ with a copy of the wild type gene. Double strand breaks (DSBs) enhance recombination and the problem of introducing a limited number of DSBs into complex genomes has been solved to a certain extent by the use of homing endonucleases. Homing endonucleases participate in intron homing in lower eukaryotes, bacteria and bacteriophages [1]. They have large target sites and generate stable long lived recombination intermediates [2, 3]. These rare cutter endonucleases, in particular the yeast dodecamer endonucleases I-Scel and the intein encoded PI-ScI have been used for inducing genome rearrangements in vertebrate cells either by introduction of purified protein or by expression from mammalian expression vectors. In Xenopus oocytes 17% of circular plasmids cleaved by I-Scel in vivo recombined with a homologous linear fragment. The same two molecules injected together did not recombine. DSBs initiated by I-Scel endonuclease at a predetermined location in the genome of mouse cells enhanced repair with a donor molecule with homologous flanking regions 100-fold more frequently than spontaneous homologous recombination [4]. Yeast I-Scel is not toxic to mouse cells and the growth of cells expressing the endonuclease constitutively is apparently normal [5]; the frequency of random occurrence of the I-Scel 18-bp recognition site is expected to be 418 (7 × 1010). However, whereas introduction of a DSB stimulated homologous recombination 100-fold, nonhomologous recombination was stimulated more than 1,000-fold [6, 7].KeywordsHomologous RecombinationZinc FingerMating TypeHoming EndonucleaseMating Type LocusThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.