The advent of recombinant DNA technology together with DNA mediated gene transfer methods permits introduction of virtually any DNA sequence into mammalian cells. Since there are a large number of human genetic diseases which are due to single gene disorders, the gene transfer systems raised the possibility of gene replacement therapy. In order for gene replacement therapy to be a reality, it is necessary to have highly efficient methods of introducing genes into primary cells. In addition it is now clear that proper regulation of several, if not all, exogenously introduced mammalian genes is dependent upon their chromosomal location. As such, methods to detect and enhance homologous recombination need to be developed before gene replacement therapy can be attempted. We propose a series of experiments which are aimed at these questions. Plasmid DNA containing selectable genes such as bacterial neomycin resistance gene or herpes viral thymidine kinase gene and non-selectable genes such as human globin genes will be used in these experiments. Homologous recombination and gene conversion will be examined by introducing deletion plasmids into mammalian cells and homologous recombination events will be selected. Parameters that affect the rate of homologous recombination will be examined. Based upon certain observations in yeast, specific methods, such as DNA double strand breaks will be tested for their enhancing affect on recombination. Legitimate recombination between chromosomal genes and their exogenously introduced counterparts will also be examined. Finally experiments to develop an in vitro system for recombination are proposed.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM033943-02
Application #
3284156
Study Section
Mammalian Genetics Study Section (MGN)
Project Start
1985-04-01
Project End
1988-03-31
Budget Start
1986-04-01
Budget End
1987-03-31
Support Year
2
Fiscal Year
1986
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
Montgomery, K T; LeBlanc, J M; Tsai, P et al. (1993) Characterization of two chromosome 12 cosmid libraries and development of STSs from cosmids mapped by FISH. Genomics 17:682-93
Yoon, S J; Seiler, S H; Kucherlapati, R et al. (1992) Organization of the human skeletal myosin heavy chain gene cluster. Proc Natl Acad Sci U S A 89:12078-82
Campbell, C; Gulati, R; Nandi, A K et al. (1991) Generation of a nested series of interstitial deletions in yeast artificial chromosomes carrying human DNA. Proc Natl Acad Sci U S A 88:5744-8
Campbell, C R; Keown, W; Lowe, L et al. (1989) Homologous recombination involving small single-stranded oligonucleotides in human cells. New Biol 1:223-7
Campbell, C R; Ayares, D; Watkins, K et al. (1989) Single-stranded DNA gaps, tails and loops are repaired in Escherichia coli. Mutat Res 211:181-8
Song, K Y; Schwartz, F; Maeda, N et al. (1987) Accurate modification of a chromosomal plasmid by homologous recombination in human cells. Proc Natl Acad Sci U S A 84:6820-4
Ayares, D; Ganea, D; Chekuri, L et al. (1987) Repair of single-stranded DNA nicks, gaps, and loops in mammalian cells. Mol Cell Biol 7:1656-62
Ganea, D; Moore, P; Chekuri, L et al. (1987) Characterization of an ATP-dependent DNA strand transferase from human cells. Mol Cell Biol 7:3124-30
Rauth, S; Song, K Y; Ayares, D et al. (1986) Transfection and homologous recombination involving single-stranded DNA substrates in mammalian cells and nuclear extracts. Proc Natl Acad Sci U S A 83:5587-91
Ayares, D; Chekuri, L; Song, K Y et al. (1986) Sequence homology requirements for intermolecular recombination in mammalian cells. Proc Natl Acad Sci U S A 83:5199-203

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