A SYSTEM UTILIZING EPSTEIN-BARR VIRUS-BASED EXPRESSION VECTORS FOR THE FUNCTIONAL CLONING OF HUMAN FIBROBLAST GROWTH-REGULATORS

The acquired ability of adherent mammalian cells to grow in suspension is closely linked to tumorigenic transformation. The anchorage-indepe ndence phenotype is likely to result from bypassing an adherence-respo nsive cell-cycle checkpoint at the G1/S boundary of the cell cycle. In order to identify genes that are part of or act upon the anchorage si gnal transduction pathway, we have developed a system which allows fun ctional cloning of regulatory genes by expression of libraries of cDNA inserts either in the sense or antisense direction. The system is com prised of two components: (i) the library expression vectors, CMV-EL a nd C1E-EL, containing EBoriP for replication in EBNA-1-expressing cell s, an expression cassette with a multiple cloning site suitable for di rectional insertion of cDNA libraries generated by standard protocols, and loxP sites which allow rapid manipulation of recovered vectors wi thout the use of restriction enzymes and (ii) the EBNA-1-producing cel l line, BB-5, a derivative of the immortalized, non-tumorigenic and an chorage-dependent human fibroblast cell line, MSU1.1. The growth chara cteristics of BB-5 cells did not differ from its parental cell line. B B-5 cells supported the episomal replication of CMV-EL and C1E-EL and allowed recovery of the vector from Hirt lysates of transfected BB-5 c ells. BB-5 cells transformed to anchorage-independent growth by transf ection with a mutant c-Ka-ras gene inserted into CMV-EL could be accur ately and efficiently identified in a background of non-transfected BB 5 cells by screening for anchorage-independent colonies with the aid o f computer-assisted image analysis.