Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1997-09-23
2001-03-13
Jones, W. Gary (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091400
Reexamination Certificate
active
06200750
ABSTRACT:
The present invention is directed to homologous recombination events and methods of identifying DNA sequences which direct the same. Methods of gene therapy are also provided in which genetic recombination is directed using DNA sequences identified in accordance with the present invention.
Recognition of cis-acting DNA signals occupies a central role in both site-specific and general recombination pathways, wherein genetic material or the sequence thereof can be exchanged from one polynucleotide to another, resulting in recombinant, i.e., new, sequences of the polynucleotide receiving the genetic material or the sequence thereof Signals in site-specific pathways define the points at which the exchange will occur. General signals do not promote defined rearrangements but rather create “hotspots” by increasing the frequency of homologous recombination in their vicinity. A well-studied general signal is the crossover hotspot instigator (chi) found in
Escherichia coli
(
E. Coli
), which functions at the inception of the recombination. Although hotspots exist in eucaryotes, specific signals, mostly nuclease sites, have only been demonstrated in yeast. The recombination hotspots observed in higher eucaryotes typically lie near regions of highly repetitive sequence and are generally observed in the context of meiosis. Shiroishi et al.,
Adv. Biophy
., 31:119-132 (1995); Lafuse,
Crit. Rev. Immunol
, 11:167-194 (1991); Smith,
Experientia
, 50:234-241 (1994). Although the existence of ubiquitous, general somatic recombination signals have been proposed, nothing comparable to chi has been functionally identified Smith,
Experientia
, 50:234-241 (1994).
SUMMERY OF THE INVENTION
In accordance with the present invention is provided a system for identifiing DNA sequences that promote homologous recombination in mammalian cells comprising a library of self-inactivating retroviral vectors containing random DNA sequence inserts.
Methods of identifying DNA sequences that promote homologous recombination in mammalian cells are also provided. Such methods comprise providing a library of self inactivating retroviral vectors containing random DNA inserts and performing at least two rounds of replication by transfecting mammalian packaging cells with the retroviral vector library. Replicated vectors are recovered and sequenced wherein sequences of DNA inserts contained in recovered vectors promote homologous recombination in mammalian cells.
Therapeutic methods are also provided in accordance with the present invention wherein a patient suffering from a DNA viral infection is administered an effective amount of a construct comprising a recombination activator linked to an inactivated viral gene.
REFERENCES:
patent: 5814479 (1998-09-01), Zhou et al.
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Dornburg Ralph
Olson Paul K.
Dechert Price & Rhoads
Jones W. Gary
Sovaya Jehanne
Thomas Jefferson University
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