Chemistry: molecular biology and microbiology – Treatment of micro-organisms or enzymes with electrical or... – Modification of viruses
Patent
1988-11-21
1993-02-23
Yarbrough, Amelia B.
Chemistry: molecular biology and microbiology
Treatment of micro-organisms or enzymes with electrical or...
Modification of viruses
435 6, 435 29, 435 691, 436501, 536 235, 935 78, 935 88, C12N 1500, C12Q 168, C07H 2100
Patent
active
051889544
DESCRIPTION:
BRIEF SUMMARY
; and Greco et al, Proc. Natl. Acad. Sci. USA (1987) 84:1565-1569. Transformation of mouse cells with plasmids comprising a viral tranfection system is described by Southern and Berg, J. Mol. Appl. Genet. (1982) 1:327-341.
SUMMARY OF THE INVENTION
Methods are provided for producing non-human cells comprising a functional human transporter capable of transporting a neurotransmitter from the outside to the inside of the cell. The cells are capable of being responsive to agonists and antagonists, as well as ancillary agents associated with the function of the neurotransmitter. Non-primate or primate cells stable in culture are transformed with human genomic DNA for expression of a functional human neurotransmitter transporter.
DESCRIPTION OF THE SPECIFIC EMBODIMENTS
Non-primate or primate cells capable of growing in culture are provided which comprise a functional primate, particularly human, neurotransmitter transporter. Methods are provided for producing these cells, as well as using the cells for investigation of neurotransmitter transport function mechanisms and screening of agonists and antagonists for the neurotransmitter.
The cell cultures of this invention will for the most part be cells derived from non-primate vertebrates, particularly rodentiae, lagomorpha, equine, bovine, porcine, etc., particularly mouse and rat cells. Cells of primate origin may also be used when appropriate. The cells will normally be other than cells associated with the brain, such as neurons, astrocytes, glial cells, or the like. Thus, the cells may be fibroblasts, leukocytes, muscle, bone, stem cells, epithelial cells, endothelial cells, etc. For the most part, the cells will be stable in culture (immortalized), so as to be grown indefinitely and expanded as needed. Cells of particular interest will be those which are easily grown, stably retain the introduced DNA, have a replication system which is not particularly error prone, and do not manifest high rates of endogenous genetic recombination. Cell lines which may find use include mouse L-M fibroblasts, L-TK.sup.- fibroblasts, L-929 fibroblasts and 3T3 fibroblasts.
The neurotransmitter transporters which may be introduced into the mouse may include transporters for serotonin, GABA, glycine, dopamine, epinephrine, norepinephrine, acetylcholine, etc.
The exogenous DNA encoding the transporter may be introduced into the host cell in a variety of ways. Conveniently, the host cell may be transformed with human genomic DNA in the presence of DNA which provides for a selection marker. Thus, human genomic DNA may be prepared, as intact chromosomes or as degraded DNA fragments of from about 5 to 100 kbp, and mixed with a plasmid or linear DNA capable of stable maintenance or integration into the genome of the host cell and carrying a selection marker. Conveniently, calcium phosphate precipitation may be employed as described in the literature. For example, the DNA may be dissolved in an appropriate phosphate buffered saline solution, about pH7, and an equal volume of 0.1-0.5M calcium chloride added drop-wise. The precipitate is incubated, followed by adding to the cells and incubating for an extended period of time, usually at least about eight hours and not more than about 24 hours. The cells are separated from the precipitate and then cultured in the presence of the selection agent.
The particular manner of selection is not critical to this invention. For the most part resistance to cytotoxic agents is convenient. Various antibiotics may be employed, but a particularly convenient combination is a neomycin resistant gene APH(3')II with G418, although with appropriate cells, methotrexate with the DHFR gene, or the like may be employed. The selection gene may be associated with a replication system, since the combination of the gene with a replication system generally provides for a larger number of transformed cells which may be selected. Various replication systems are available as derivatives of SV40, bovine papilloma virus, adenovirus, etc. These vectors find ample descrip
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This invention was made with Government support under Grant Nos. EY02423 and EY02608 awarded by the National Institutes of Health/National Eye Institute. The Government has certain rights in this invention.
Chang Albert S.
Lams Dominic M.
Baylor College of Medicine
Marschel Ardin H.
Yarbrough Amelia B.
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