The Salk Institute for Biological Studies

Siska Diagnostics, Inc.

Dana-Farber-Cancer Institute Inc.

Salk Institute for Biological Sciences

Geoffrey M Wahl

Stephen V O'Gorman

Noriaki Shimizu

Soumitra S Ghosh

Emil T Kaiser

Leslie E Orgel

Gary F Musso

H Michael Shepard

Loren D Walensky

Federico Bernal

Mark Wade

Teru Kanda

Compositions and methods for increasing p53-dependent transcriptional activity in a cell.

Cancer therapies and diagnostics

Site-specific recombinases provide a means of efficiently manipulating chromosomal sequences in mammalian cells in culture and in mice. Embryonic stem cells containing recombinase nucleic acid constructs that were expressed in the male germline would simplify current protocols for producing mice bearing homologously recombined alleles that have been secondarily rearranged by a site-specific recombinase, five lines of transgenic mice containing a fusion gene consisting of the mouse protamine 1 gene promoter and the Cre recombinase coding sequence (ProCre nucleic acid constructs) showed high levels of Cre-mediated recombination of the germline, but did not show appreciable recombination in other tissues. In different ProCre strains, between 80% and 100% of the progeny that inherited a Cre target nucleic acid construct from males that were also heterozygous for a ProCre nucleic acid construct inherited the Cre-recombined target. ProCre nucleic acid constructs and recombined targets segregated in the first generation. When ES cells prepared from one ProCre line were transfected with vectors containing a loxP-flanked neomycin cassette, G418 resistant, homologously recombined clones, in which the loxP sites remained functional, were readily isolated. These data establish that ProCre nucleic acid constructs will facilitate the production of subtle, conditional, or tissue-specific mutations in mice as well as the production and analysis of mice with recombinase-conditional lethal alleles.

Site-specific recombination in eukaryotes and constructs useful therefor

The present invention relates to compositions and methods for the generation of recombinant plants by manipulation of chromosomal sequences in plant cells via recombination promoted by recombinases. Nucleic acid constructs are provided in which a germline-specific promoter is operatively associated with a recombinase coding sequence. Methods are provided in which the constructs are introduced into a variety of plant species.

A gene activation/inactivation and site-specific integration system has been developed for mammalian cells. The invention system is based on the recombination of transfected sequences by FLP, a recombinase derived from. In several cell lines, FLP has been shown to rapidly and precisely recombine copies of its specific target sequence. For example, a chromosomally integrated, silent &#x3b2;-galactosidase reporter gene was activated for expression by FLP-mediated removal of intervening sequences to generate clones of marked cells. Alternatively, the reverse reaction can be used to target transfected DNA to specific chromosomal sites. These results demonstrate that FLP can be used, for example, to mosaically activate or inactivate transgenes for a variety of therapeutic purposes, as well as for analysis of vertebrate development. The FLP recombination system of the present invention can be incorporated in transgenic, non-human mammals to achieve site-specific integration of transgenes, to construct functional genes or to disrupt existing genes.

FLP-mediated gene modification in mammalian cells, and compositions and cells useful therefor

Site-specific recombinases provide a means of efficiently manipulating chromosomal sequences in mammalian cells in culture and in animals. Embryonic stem cells containing recombinase nucleic acid constructs that were expressed in the male germline simplify current protocols for producing mice bearing homologously recombined alleles that have been secondarily rearranged by a site-specific recombinase. In different ProCre strains, between 80% and 100% of the progeny that inherited a Cre target nucleic acid construct from males that were also heterozygous for a ProCre nucleic acid construct inherited the Cre-recombined target. ProCre nucleic acid constructs and recombined targets segregated in the first generation. When ES cells prepared from one ProCre line were transfected with vectors containing a loxP-flanked neomycin cassette, G418 resistant, homologously recombined clones, in which the loxP sites remained functional, were readily isolated.

A gene activation/inactivation and site-specific integration system has been developed for mammalian cells. The invention system is based on the recombination of transfected sequences by FLP, a recombinase derived from Saccharomyces. In several cell lines, FLP has been shown to rapidly and precisely recombine copies of its specific target sequence. For example, a chromosomally integrated, silent &#x3b2;-galactosidase reporter gene was activated for expression by FLP-mediated removal of intervening sequences to generate clones of marked cells. Alternatively, the reverse reaction can be used to target transfected DNA to specific chromosomal sites. These results demonstrate that FLP can be used, for example, to mosaically activate or inactivate transgenes for a variety of therapeutic purposes, as well as for analysis of vertebrate development. The FLP recombination system of the present invention can be incorporated in transgenic, non-human mammals to achieve site-specific integration of transgenes, to construct functional genes or to disrupt existing genes.

This invention provides methods by which test substances can be screened for their ability to inhibit, enhance or eliminate double minute (DM) or extrachromosomal DNA by micronucleation in cells. This invention also provides a method for inducing maturation or death of a cell having the capacity to generate micronuclei. It also provides a method of treating a disease in a subject, the cells correlated with the disease having DM and extrachromosomal DNA as well as the capacity to generate micronuclei to capture them. Further provided is a method of detecting chromosomal and extrachromosomal DNA in a cell.

Compositions and methods for treating cells having double minute DNA

The present invention provides a method for isolating nucleic acid from micronuclei separated from a cell that involves separating micronuclei from a cell and isolating nucleic acid from the micronuclei.

Method for isolating nucleic acid from micronuclei separated from a cell

The present invention provides a method for the isolation of extrachromosomal amplified nucleic acids that are associated with a cell proliferative disorder. Isolation and further identification of such genes is critical for diagnosis, prognosis, and course of therapy.

Method for isolation of extrachromosomal amplified genes

A gene activation/inactivation and site-specific integration system has been developed for mammalian cells. The invention system is based on the recombination of transfected sequences by FLP, a recombinase derived from Saccharomyces. In several cell lines, FLP has been shown to rapidly and precisely recombine copies of its specific target sequence. For example, a chromosomally integrated, silent .beta.-galactosidase reporter gene was activated for expression by FLP-mediated removal of intervening sequences to generate clones of marked cells. Alternatively, the reverse reaction can be used to target transfected DNA to specific chromosomal sites. These results demonstrate that FLP can be used, for example, to mosaically activate or inactivate transgenes for a variety of therapeutic purposes, as well as for analysis of vertebriate development.

FLP-mediated gene modification in mammalian cells, and compositions and

Fluorescent linker moieties are provided which comprise a fluorescent compound such as fluorescein attached to a linker moiety such that a functional group of the linker is available for attachment to an affinity molecule such as a nucleic acid which has an N.sup.4 (substituted amino) cytosine moiety. Probes tagged with fluorescent derivatives such as fluorescein, tetramethyrhodamine or tetraethylrhodamine may be detected by fluorescence spectroscopic methods.

Fluorescent compounds for tagging nucleic acid probes

Nucleic acid hybridization probes are provided which comprise an N.sup.4 -(substituted amino)cytosine moiety, wherein the substituted amino group comprises a tag moiety, whereby the probe is detected. Methods of preparing probes of the invention, intermediates used in such methods, and methods of using the probes of the invention in hybridization assays are also provided. Typical tag moieties employed with the probes of the invention are biotinyl, aminothiadiazole and fluorescein derivatives, connected to N.sup.4 -amino groups of modified cytosines of the probe through linker moieties. Probes tagged with biotin are typically detected by binding to the biotinyl moieties, through a streptavidin or avidin molecule, a reporter group which includes streptavidin or avidin and then detecting a signal due to the reporter group. Probes tagged with aminothiadiazole derivatives are typically detected by essentially the same method as those tagged with biotinyl but employing as reporter group one which binds to the derivative through a carbonic anhydrase molecule. Probes tagged with fluorescein derivatives are detected by a fluorescence spectroscopic method without binding of a reporter group to the tag.

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San Diego, CA, United States of America

Geoffrey M Wahl