Transformation of mammalian cells with an amplifiable dominant-acting gene.

Abstract

We have transferred a mutant hamster gene coding for an altered dihydrofolate reductase to wild-type cultured mouse cells by using total genomic DNA from methotrexate-resistant Chinese hamster ovary A29 cells as donor. By demonstrating the presence of hamster gene sequences in transformants we have provided direct evidence for gene transfer. Transformants selected for increased resistance to methotrexate contain increased amounts of the newly transferred gene. We have used this mutant dhfr gene to introduce the Escherichia coli antibiotic resistance plasmid pBR322 into animal cells. Amplification of the dhfr sequences results in amplification of the pBR322 sequences as well. The use of this gene may allow the introduction and amplification of virtually any genetic element in various new cellular environments. The ability to transfer purified genes into cultured cells provides a unique opportunity to study the function and physical state of exogenous genes in new cellular environments. The development of systems for DNA transfer in animal cells originated with the lytic transfection of cells by using purified viral DNA (1, 2) and progressed to the stable transfer of viral transforming functions to appropriate recipient cells (3). Subsequently, viral genes from the herpesviruses coding for the biochemically selectable marker thymidine kinase (TK) (4-6) were transferred to enzyme-deficient mutant cells. Restriction fragments of herpes simplex virus type 1 encoding TK were isolated (6) and subsequently cloned into bacterial plasmids (7). Through the use of this selectable marker, virtually any gene can now be introduced into recipient cells (8, 9); however, these cells must be tk-mutants. Other potential selection systems are available, and several laboratories have recently demonstrated the DNA-mediated transfer of cellular genes coding for selectable markers such as TK (10), adenine phosphoribosyltransferase (11) and hypoxanthine phosphoribosyltransferase (12,13 Transformation and Selection. Ltk-aprF cells and NIH 3T3 cells were transformed with genomic DNA by the calcium phosphate coprecipitation method (2) as described (11). All DNAs were sterilized by ethanol precipitation and resuspended in 1 mM Tris-HCl/1 mM EDTA, pH 7.9. For tk+ transformation, cells were exposed to hypoxanthine/aminopterin/thymidine selective medium as described (10). Transformants resistant to Mtx were select~d in growth medium containing either 0.1 or 0.2 jqg of Mtx per ml with the same feeding schedule as for tk selection. Afte 2-3 weeks, colonies were isolated from individual dishes with cloning cylinders to ensure that each transformant arose from an independent event. In transformation with ligated DNAs, no more than 1 ,ug of pBR322 DNA was added to 106 cells per dish because higherThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.3567Abbreviations: DHFR, dihydrofolat...

Adeno-associated virus vectors can be efficiently produced without helper virus

“…Surprisingly, cultures cotransfected with pJM17 and cultures infected with adenovirus-2 produced similar amounts of AAV lacZ vector. pJM17 does produce extremely low levels of adenovirus when transfected into 293 cells in the presence or absence of AAV vector and helper plasmids (typically 5 × 10 6 cells transfected with 10 g of pJM17 by the calcium phosphate method 24 produce a total of 10 4 plaque forming units (p.f.u.)). This is due to an infrequent rearrangement of the circular genome that occurs in a small number of transfected cells.…”

Convection-Enhanced Delivery of AAV Vector in Parkinsonian Monkeys; In Vivo Detection of Gene Expression and Restoration of Dopaminergic Function Using Pro-drug Approach

“…The expression casette contained a CMV promoter, a chimeric intron composed of a CMV splice donor and a human globin splice acceptor site, human growth hormone polyadenylation sequence, and flanking AAV ITRs (inverted terminal repeats) (39).…”

DNA‐mediated gene transfer as an indicator of DNA damage and its repair by recipient cells

“…For plasmid modification, "C-labelled MMS or MNU (Amersham, Bucks, England) in DMSO solution at the indicated concentrations were reacted with 2 pg of plasmid pHSV106 DNA [7,8] Mouse LTK-/aprtcells [9], maintained as monolayers in Dulbecco's modified Eagle's medium (Gibco) with 10% calf serum and antibiotics, were routinely passaged 1:3 once per week. 4 x lo5 mouse LTK-cells were seeded 24 h before transformation and transfections were carried out using the calcium phosphate-DNA coprecipitation technique [ 11.…”