A general strategy has been developed for expression and rescue of cDNAs in mammalian cells. cDNA libraries are constructed in a new phage vector, λPMV, which contains simian virus 40 (SV40) early region promoter sequences for transcription of cDNA inserts, as well as a dominant-acting selectable marker neo. Efficient transfer of the cDNA library to mammalian cells can be achieved by phage particle-mediated transfection. Following selection in the antibiotic G418, cells expressing the phenotype of interest are identified and isolated. Rapid recovery of the transfected cDNA is achieved through cell fusion of the transduced cells with COS cells. Replication at the SV40 origin promotes excision of the integrated cDNA as small circular DNA, which after isolation in this form is used to transform bacteria to ampicillin resistance. To test this strategy, a cDNA encoding the human lymphocyte differentiation antigen CD8 was inserted into λPMV. CD8 expression on the surface of mouse L cells and the efficient recovery of full-length CD8 cDNA inserts confirm the feasibility of this system. It is anticipated that the single-step screening of libraries constructed in λPMV will allow for the isolation of rare cDNAs and will prove less laborious than methods currently available.
ASJC Scopus subject areas
- Molecular Biology