Homologous recombination between mammalian cellular sequences and their counterparts introduced by DNA transfer methods could play an important role in human gene therapy. It can also be used for gene-knockout which will provide a new genetic tool for analysis of gene function. Homologous recombination between input plasmid sequences and between input plasmid and chromosomal sequences has been demonstrated. In order for these methods to be of general utility it is necessary to enhance the frequencies of homologous recombination. This proposal is aimed at developing methods to achieve gene replacement and insertional inactivation (gene knockout) of mammalian genes by homologous recombination. Autonomously replicating vectors will be used to achieve extremely high frequencies of homologous recombination. A method to achieve hit-and-run recombination to modify cellular genes is proposed. Use of replicating and non-replicating vectors to achieve gene knockout are also proposed. A particular problem with mammalian cells is that not one but two copies of a gene have to be inactivated to observe a recessive phenotype. Use of double knock-out vectors and gene fusions are proposed to achieve this goal. Examination of repair of heteroduplexes, which are generated during the recombination process, in normal human cells and cells from patients who exhibit chromosomal fragility syndromes is proposed. Repair of single base mismatches, clustered base mismatches, single strand gaps and loops will be examined by using heteroduplexes prepared from autonomously replicating plasmids. Finally, a series of experiments aimed at developing a cell-free system to study repair of heteroduplexes is also proposed. Many of the experiments are based on previous successes in the applicant's laboratory.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM033943-04
Application #
3284155
Study Section
Molecular Cytology Study Section (CTY)
Project Start
1985-04-01
Project End
1993-03-31
Budget Start
1988-04-01
Budget End
1989-03-31
Support Year
4
Fiscal Year
1988
Total Cost
Indirect Cost
Name
University of Illinois at Chicago
Department
Type
Overall Medical
DUNS #
121911077
City
Chicago
State
IL
Country
United States
Zip Code
60612
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