Angel Pellicer

Transfer of purified herpes virus thymidine kinase gene to cultured mouse cells.

Treatment of Ltk−, mouse L cells deficient in thymidine kinase (tk), with Bam I restriction endonuclease cleaved DNA from herpes simplex virus-1 (HSV-1) produced tk+ clones with a frequency of 10−6/2 μg of HSV-1 DNA. Untreated cells or cells treated with Eco RI restriction endonuclease fragments produced no tk+ clones under the same conditions. The thymidine kinase activities of four independently derived clones were characterized by biochemical and serological techniques. By these criteria, the tk activities were found to be identical to HSV-1 tk and different from host wildtype tk. The tk+ phenotype was stable over several hundred cell generations, although the rate of reversion to the tk− phenotype, as judged by cloning efficiency in the presence of bromodeoxyuridine, was high (1–5 × 10−3). HSV-1 DNA Bam restriction fragments were separated by gel electrophoresis, and virtually all activity, as assayed by transfection, was found to reside in a 3.4 kb fragment. Transformation efficiency with the isolated fragment is 20 fold higher per gene equivalent than with the unfractionated total Bam digest. These results prove the usefulness of transfection assays as a means for the bioassay and isolation of restriction fragments carrying specific genetic information. Cells expressing HSV-1 tk may also provide a useful model system for the detailed analysis of eucaryotic and viral gene regulation.

Transformation of mammalian cells with genes from procaryotes and eucaryotes

Abstract We have stably transformed mammalian cells with precisely defined procaryotic and eucaryotic genes for which no selective criteria exist. The addition of a purified viral thymidine kinase (tk) gene to mouse cells lacking this enzyme results in the appearance of stable transformants which can be selected by their ability to grow in HAT. These biochemical transformants may represent a subpopulation of competent cells which are likely to integrate other unlinked genes at frequencies higher than the general population. Co-transformation experiments were therefore performed with the viral tk gene and bacteriophage ΦX174, plasmid pBR322 or the cloned chromosomal rabbit β-globin gene sequences. Tk + transformants were cloned and analyzed for co-transfer of additional DNA sequences by blot hybridization. In this manner, we have identified mouse cell lines which contain multiple copies of 4)X, pBR322 and the rabbit β-globin gene sequences. The ΦX co-transformants were studied in greatest detail. The frequency of co-transformation is high: 15 of 16 tk + transformants contain the ΦX sequences. Selective pressure was required to identify co-transformants. From one to more than fifty ΦX sequences are integrated into high molecular weight nuclear DNA isolated from independent clones. Analysis of subclones demonstrates that the ΦX genotype is stable through many generations in culture. This co-transformation system should allow the introduction and stable integration of virtually any defined gene into cultured cells. Ligation to either viral vectors or selectable biochemical markers is not required.

Isolation of transforming DNA: Cloning the hamster aprt gene

We have isolated the hamster gene coding for the enzyme adenine phosphoribosyl transferase (aprt) using gene transfer and molecular cloning of transforming DNA. Mouse aprt- cells were transformed to the aprt+ phenotype with the product of ligation of Hind III-cleaved hamster genomic DNA and pBR322 DNA. In this manner, the aprt gene was linked to a marked plasmid sequence and segregated from other hamster sequences. A lambda-recombinant phage containing pBR322 DNA sequences was isolated from a library of aprt+ transformed cell DNA. The phage DNA transfers hamster aprt+ activity at a frequency expected of a pure gene. Furthermore, sequences homologous to this clone are present in all hamster aprt+ transformants examined. This experimental design should in theory permit the isolation of any gene coding for selectable or identifiable functions for which DNA-mediated gene transfer can be effected.

Transformation of Mammalian Cells

The introduction of foreign DNA into cells can result in a stable and heritable change in phenotype. The ability to transfer purified genes provides the unique opportunity to study the function and physical state of exogenous genes in the transformed host and extends the powerful methods of virus genetics to cellular genetics. The use of transformation* falls into three categories: 1) transformation as a means for gene purification; 2) transformation as a way of studying the structure and function of purified genes; 3) transformation as a tool for dissecting complex phenotypes. This paper is concerned with the development of transformation in cultured mammalian cells. As this is a very young field, we shall draw mainly on our own work.