Recent advances in gene transfer have made possible the introduction of genetic material into intact animals or their bone marrow cells. One goal of these experiments is to insert altered dihydrofolate reductase (DHFR) genes into bone marrow, thereby rendering it resistant to methotrexate (MTX). This would allow treatment of malignancies with high doses of MTX without fear of bone marrow toxicity. If such a system worked it could be applied to human beings. The problem with experiments attempted thus far is that gene transfer protocols are not effective enough to assure that the bone marrow will be uniformly transformed with an efficiently expressed MTX-resistant DHFR gene. Thus, the applicability of this approach to humans remains to be demonstrated. The purpose of our experiments is to establish a system wherein the chances of inducing host resistance to MTX are optimized. We have produced a number of lines of transgenic mice by microinjecting a mutant DHFR gene into one-celled embryos. This gene specifies a product that is highly resistant to MTX. All animals have the new gene integrated into the germ line and transmit it to progeny, which now carry it in every cell. Several lines also express the gene in several somatic tissues including bone marrow. We will administer MTX to progeny and control littermates not carrying the DHFR gene in order to select in vivo for resistance to the drug. When these mice develop MTX resistance, we will cross them to inbred animals and innoculate the transgenic progeny with malignant cells. We will then give MTX to determine if cancer can be cured in the genetically MTX-resistant transgenic mice. We also propose to improve the model for isolated bone marrow resistance. Our approach entails the production of additional transgenic mice on the inbred C3H background. After the animals have been selected for bone marrow resistance we will transplant their bone marrow to irradiated C3H recipients. These latter animals will thus receive bone marrow cells which are uniformly transformed with the MTX-resistant DHFR gene and which are known to express the gene efficiently. The response of these animals to MTX therapy should provide definitive information regarding the feasibility of gene transfer as an adjunct to chemotherapy.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA042103-02
Application #
3182944
Study Section
Experimental Therapeutics Subcommittee 2 (ET)
Project Start
1986-09-30
Project End
1990-02-28
Budget Start
1988-03-01
Budget End
1989-02-28
Support Year
2
Fiscal Year
1988
Total Cost
Indirect Cost
Name
Mount Sinai School of Medicine
Department
Type
Schools of Medicine
DUNS #
City
New York
State
NY
Country
United States
Zip Code
10029
Gordon, J W (1993) Micromanipulation of gametes and embryos. Methods Enzymol 225:207-38
Gordon, J W (1993) Production of transgenic mice. Methods Enzymol 225:747-71
Reventos, J; Sullivan, P M; Joseph, D R et al. (1993) Tissue-specific expression of the rat androgen-binding protein/sex hormone-binding globulin gene in transgenic mice. Mol Cell Endocrinol 96:69-73
Mangues, R; Seidman, I; Gordon, J W et al. (1992) Overexpression of the N-ras proto-oncogene, not somatic mutational activation, associated with malignant tumors in transgenic mice. Oncogene 7:2073-6
Isola, L M; Gordon, J W (1991) Transgenic animals: a new era in developmental biology and medicine. Biotechnology 16:3-20
Mangues, R; Seidman, I; Pellicer, A et al. (1990) Tumorigenesis and male sterility in transgenic mice expressing a MMTV/N-ras oncogene. Oncogene 5:1491-7
Greenstein, R J; Isola, L; Gordon, J (1990) Differential cholecystokinin gene expression in brain and gut of the fasted rat. Am J Med Sci 299:32-7
Gordon, J W (1990) Micromanipulation of embryos and germ cells: an approach to gene therapy? Am J Med Genet 35:206-14
Bradbury, M W; Isola, L M; Gordon, J W (1990) Enzymatic amplification of a Y chromosome repeat in a single blastomere allows identification of the sex of preimplantation mouse embryos. Proc Natl Acad Sci U S A 87:4053-7
Gordon, J W; Uehlinger, J; Dayani, N et al. (1990) Analysis of the hotfoot (ho) locus by creation of an insertional mutation in a transgenic mouse. Dev Biol 137:349-58

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