Mouse thymidine-kinase-negative L cells were transformed with a cloned chicken ovalbumin gene linked to the cloned thymidine kinase gene from herpes simplex virus type 1. Most thymidine-kinase-positive clones contained one or more copies of the ovalbumin gene, which were stably maintained for at least 6 months during continuous culture under selective conditions. Transcription of the ovalbumin gene was detected at a low level, producing RNA molecules that were correctly spliced and polyadenylylated by comparison with genuine ovalbumin mRNA. However, the 5' end of these RNA molecules does not correspond to that of ovalbumin mRNA. Molecular cloning has made possible the determination of the structure of the chicken ovalbumin gene (1), which is expressed under hormonal control in the oviduct tubular gland cells (2). By comparison with the structure of other genes, we have identified several conserved sequences that may play a role in the regulation of the expression of the ovalbumin gene and in the splicing of its primary transcript. However, for the true significance of these sequences to be evaluated, they, or mutants derived from them, must be tested in appropriate in vitro or in vivo systems (3, 4). We describe here our first approach to the development of such an in vivo biological system, which consists of introducing the ovalbumin gene into a heterologous cultured eukaryotic cell. Previous work (5-7) has indeed shown that it is possible to introduce foreign DNA into thymidinekinase-negative (TK-) mouse L cells by cotransformation with a thymidine kinase gene from herpes simplex virus type 1. Using this approach, we have introduced the chicken ovalbumin gene into L cells.
MATERIALS AND METHODSPlasmids and Enzymes. pBovl and pBov2 were constructed as described in the legends to Figs. la and 3b and in the text. Other plasmids and enzymes have been described elsewhere (8-12). Ovalbumin mRNA (ov-mRNA) was a gift from M. LeMeur and A. Krust. Cell Culture and Transformation. LMTK-cells were grown in monolayer cultures as described (5). LMTK+ cells were maintained in the presence of hypoxanthine/aminopterin/ thymidine medium. Six micrograms of pBovl and 1 ,g of TK-M4 plasmid DNA (7,8,13) were cleaved at their single Sal I sites, extracted with phenol, and precipitated with ethanol.They were then ligated with Biolabs T4 DNA ligase (1 Al) in a volume of 50 Ml for 3 hr before phenol extraction and ethanol precipitation. The heterologous concatenates were used to transform LMTK-cells on five 100-mm petri dishes as described (5). The TK+ phenotype was selected as described (5), and 10 isolated colonies of cells were picked by using the sterile cylinder technique.Analysis of DNA. DNA was isolated from confluent cultures. After digestion with proteinase K (100 ,g/ml), phenol/chloroform extraction, and ethanol precipitation, the DNA was digested with a restriction enzyme by using adenovirus DNA as digestion marker (10). Electrophoresis, blotting, and hybridization to nick-translation probes were as described (10 (...