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-independence phenotype is likely to result from bypassing an adherence-responsive 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 signal transduction pathway, we have developed a system which allows functional cloning of regulatory genes by expression of libraries of cDNA inserts either in the sense or antisense direction. The system is comprised of two components: (i) the library expression vectors, CMV-EL and C1E-EL, containing EBoriP for replication in EBNA-1-expressing cells, an expression cassette with a multiple cloning site suitable for directional insertion of cDNA libraries generated by standard protocols, and loxP sites which allow rapid manipulation of recovered vectors without the use of restriction enzymes and (ii) the EBNA-1-producing cell line, BB-5, a derivative of the immortalized, non-tumorigenic and anchorage-dependent human fibroblast cell line, MSU1.1. The growth characteristics of BB-5 cells did not differ from its parental cell line. BB-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 cells. BB-5 cells transformed to anchorage-independent growth by transfection with a mutant c-Ka-ras gene inserted into CMV-EL could be accurately and efficiently identified in a background of non-transfected BB5 cells by screening for anchorage-independent colonies with the aid of computer-assisted image analysis.