Isolation of a homogeneous pool of cells expressing a particular gene is an important step in the study of that gene's function. As gene transfer technology has progressed, the need for homogenous pools of cells expressing genes of interest remains. New restrictions as to the potential immunogenicity and the efficiency of stable cell selection call for new technology to be developed. The project proposed here is aimed toward developing an inducible system whereby a homogeneous pool of transfected cells can be selected from culture in a nondestructive manner without the use of antibiotic selection. A selection system that inducibly expresses a molecular tag on the surface of transfected cells will be generated with the goal being clearance of the tag from the cell membrane after isolation of those cells. Upon successful development of this system, researchers will be able to generate homogeneous pools of transfected cells lacking membrane tags without using antibiotic selection. This type of system would immediately lend itself to many applications where a membrane tag would interfere, such as experimentation with recombinant membrane proteins. Since the pool of selected cells would not display recombinant membrane tags, these cells could be introduced into animals without potential immunologic consequences.
The commercialization of vectors resulting in the Phase I development of this project will allow researchers to select transfected cells from culture rapidly and without the use of antibiotic selection. This selection will render a homogeneous pool of cells that do not display recombinant membrane selection tags. These transfected cells will be useful for studies involving recombinant mutant and wild type membrane proteins without interference from the membrane tag. Furthermore, by using this system, pools of cells expressing various recombinant proteins such as signaling molecules and transcription factors could be introduced into animal models for in vivo studies.
