- Authors: Melidoni, A. N., Dyson, M. R., McCafferty, J.
- Year: 2016
- Journal: Methods Mol Biol 1341 111-32
- Applications: in vitro / DNA / PEIpro
- Cell type: HEK-293-F
Description: Human embryonic kidney Fibroblast
Known as: FreeStyle 293-F, HEK293-F, 293-F
HEK-293F suspension cells (50 mL at 10^6 cells/mL) were transfected with 50 µg DNA and 100 µL PEIpro. Culture supernatants were harvested 5 days post-transfection.
Antibodies able to bind and modify the function of cell surface signaling components in vivo are increasingly being used as therapeutic drugs. The identification of such "functional" antibodies from within large antibody pools is, therefore, the subject of intense research. Here we describe a novel cell-based expression and reporting system for the identification of functional antibodies from antigen-binding populations preselected with phage display. The system involves inducible expression of the antibody gene population from the Rosa-26 locus of embryonic stem (ES) cells, followed by secretion of the antibodies during ES cell differentiation. Target antigens are cell-surface signaling components (receptors or ligands) with a known effect on the direction of cell differentiation (FGFR1 mediating ES cell exit from self renewal in this particular protocol). Therefore, inhibition or activation of these components by functional antibodies in a few elite clones causes a shift in the differentiation outcomes of these clones, leading to their phenotypic selection. Functional antibody genes are then recovered from positive clones and used to produce the purified antibodies, which can be tested for their ability to affect cell fates exogenously. Identified functional antibody genes can be further introduced in different stem cell types. Inducible expression of functional antibodies has a temporally controlled protein-knockdown capability, which can be used to study the unknown role of the signaling pathway in different developmental contexts. Moreover, it provides a means for control of stem cell differentiation with potential in vivo applications.