Multicellular living organisms and unmodified parts thereof and – Method of using a transgenic nonhuman animal in an in vivo...
Reexamination Certificate
1998-07-17
2003-05-20
Priebe, Scott D. (Department: 1632)
Multicellular living organisms and unmodified parts thereof and
Method of using a transgenic nonhuman animal in an in vivo...
C800S013000, C800S018000, C800S021000, C800S024000, C800S025000, C435S325000, C435S352000, C435S354000, C435S455000, C435S463000
Reexamination Certificate
active
06566579
ABSTRACT:
BACKGROUND OF THE INVENTION
The ability to introduce specific alterations of endogenous genes into the germline of mice via targeted mutagenesis in embryonic stem cells (ES) has represented a major breakthrough in mouse genetics. Thus gene inactivation has been widely used to examine the effect of loss of function in various biological processes and has already permitted to accumulate a wealth of new insights into gene function and also to create mouse models of human genetic diseases. However it would also be useful to introduce subtle mutations, in order to refine the genetic analysis and to better approximate the models of genetic diseases which do not necessarily result from null mutations. Thus several strategies have been developped, aimed at generating subtle mutations in a given gene. However, one common limitation to all the current gene targeting procedures is the low frequency of correct targeting, which becomes a serious problems especially when using two successive rounds of targeting. Therefore, efforts have been made to increase the efficiency of gene targeting by several means like increasing the size of the region of homologies with the target locus, using isogenic genomic DNA, or improving the selection procedures.
SUMMARY OF THE INVENTION
In this paper, we present an alternative approach, to overcome these limitations, which relies on the observation that double-strand ends of broken chromosomes are highly recombinogenic; we reasoned that the introduction of a double stranded break (DSB) in an endogenous gene could increase targeting frequency through stimulation of the cellular recombination machinery. Thus our approach which involves two steps, is based on the induction of a specific DSB in a natural locus, the gene encoding villin, an actin binding protein mainly expressed in the intestine and kidney (louvard). In a first step, an I-Sce I restriction site is introduced into the villin locus. Indeed, I-Sce I, a very rare cutter endonuclease has been shown to initiate DSB in the mammalian genome, while its random integration allowed new insights into the analysis of recombination mechanisms in mammals. In the second step, the effect of DSB on DNA repair and homologous recombination frequency is assessed after cotransfer of an I-Sce I expressing vector and of a villin replacement vector in the targeted ES cells (FIG.
1
). We found that the presence of a DSB in the target gene not only enhanced homologous replacement by the incoming DNA but also induced gene conversion at a very high rate.
REFERENCES:
patent: 5474896 (1995-12-01), Dujon et al.
patent: 5830729 (1998-11-01), Jaisser et al.
patent: 0 206 846 (1986-12-01), None
patent: 0 206 849 (1986-12-01), None
patent: 0 367 666 (1990-05-01), None
patent: 0 419 621 (1991-04-01), None
patent: 0 682 111 (1995-11-01), None
patent: 0 682 112 (1995-11-01), None
Rousseau-Merck, et al., Human Genetics, vol. 78, pp. 130-133, 1988.*
Rousseau-Merck, et al., Human Genetics, vol. 78, pp. 130-133, 1988.*
Moreadith et al., Journal of Molecular Medicine, vol. 75, pp. 208-216., 1997.*
Hammer et al., Journal of Animal Science, vol. 63, pp. 269-278., 1986.
Babinet Charles
Choulika Andre
Cohen-Tannoudji Michel
Jaisser Frederic
Louvard Daniel
Finnegan Henderson Farabow Garrett & Dunner L.L.P.
Institut Pasteur
Priebe Scott D.
Woitach Joseph
LandOfFree
I-Sce I induced gene replacement and gene conversion in... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with I-Sce I induced gene replacement and gene conversion in..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and I-Sce I induced gene replacement and gene conversion in... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3008155