Work Package 6

Generation of single chain antibodies to signaling molecules, transcription factors and chromatin modulators of stem and differentiated cells

Introduction
Monoclonal antibodies are powerful tools that have found wide applications in the life sciences, the clinic and industry. The ability to identify, enrich and functionally manipulate stem cells and their differentiated progeny has been facilitated by use of monoclonal antibodies. However, conventional monoclonal antibodies are often not optimal due to isotype or low affinity, and the fact that they consist of two polypeptide chains encoded by separate genes. Recent molecular technology has allowed the generation of synthetic and/or natural repertoires of antibody specificities in the form of single chain monoclonal antibody libraries adapted for easy growth and selection into a phage genome. The fact that Camelids (such as Llamas) naturally produce single chain antibodies has allowed the generation of phage libraries encoding the single chain immunoglobulin repertoire from B-cells of immunized animals. Such single chain antibodies are stable, soluble, of high affinity and the genes encoding these antibodies can easily be manipulated for a wide range of downstream applications.For our studies on signaling molecules in stem cells (ES cells, hematopoietic stem cells, Schwann cell precursors) and differentiated cells types (ES derived myogenic cells, erythroid precursors, Schwann cells), single chain monoclonal antibodies are advantageous and and will allow for 1.) identification of novel signaling molecules; 2.) diagnostics on cell preparations. For example, to ensure that all cells in a differentiated ES cell culture are indeed differentiated and that no undifferentiated cells remain); 3) purification procedures to isolate stem and/or differentiated cells; 4) immunohistology to localize stem cells/differentiated cells in vivo; 5) functional blocking studies to test the role of the specific molecule in the cell.

Objectives
1.) To generate and select single chain monoclonal antibodies specific for important signaling molecules, transcription factors and chromatin modulators identified in our studies. 2.) To use these single chain antibodies as diagnostic tools, in novel signaling molecule discovery, in affinity purification of antigen containing multi-protein complexes, to specifically inhibit signaling cascades, in the study of normal protein function of the specified antigen bearing molecule and for cell enrichment and in vivo localization studies.

Approach
To obtain single chain monoclonal antibodies specific for the signaling molecules and transcription factors under study single chain antibody libraries will be generated from synthetic libraries or from B-cells of Llamas immunized with cocktails (containing tens of different antigenic peptides) of bacterially over-expressed antigens. The choice of antigens will be determined by the outcome of several ongoing studies and will include transcription factors such as Oct-6, Brn-2, Gata-1, Ldb1, Brahma and EKLF and may include stem and differentiated cells. Single chain antibody libraries will be positively selected on the listed factors, stem cells and/or differentiated progeny including ES cells, hematopoietic stem cells and Schwann cell precursors. Selected phage clones will be further manipulated for various downstream applications.

Link with other projects: This is a highly technology driven project with obvious links to all other projects, in particular WP2, 5, 8, 9 and 10 Keywords: single chain antibodies, signaling, transcription factors,stem cells, diagnostic markers

Participants: Dzierzak, Mummery, Grosveld, Meijer, van Lohuizen, Verrijzer,