CORE--GENOMICS AND PROTEOMIC RESOURCES

Wistar Institute, Philadelphia PA

Investigators

Linked publications & trials

Abstract

DESCRIPTION (provided by applicant): Core D fulfills the need of many Project and Pilot investigators to follow-up on the global screening analysis and characterize the target genes and proteins that have been selected for their differential expression in cancers of the skin. Three of the 5 projects and 4 of the 6 Pilot Studies will use global screens to identify new genes related to cancers of the skin. Gene identification will be done at the mRNA (Projects 3, 4, and 5 and Pilot Studies 2, 3, and 4) or DNA (Pilot Studies 1 and 2) levels. In many cases, we will only identify gene fragments (EST). As a first step, we will need to identify and clone full-length cDNA. The characterization and cloning of cDNA will be done in core Component 1, managed by Dr. Arupa Ganguly. Core Component 2 under the direction of Dr. William Wunner will produce recombinant baculoviruses and will express the proteins of interest. The facility will recommend a plasmid transfer vector(s) that will permit identification and purification of the expressed novel recombinant proteins. Recombinant baculoviruses will be produced in the facility by co-transfection of the transfer vector and appropriate baculovirus (Bac-N-BlueTM) genome DNA in Spodoptera frugiperda (Sf9) insect cells. Recombinant virus will be amplified in Sf9 cells and the recombinant proteins (the cell-associated proteins and secretory proteins with native secretion signals selected in the gene screen) will be expressed and produced to high level in Sf9 or Trichoplusia in (High Five) insect cells. Core Component 3, under the direction of Dr. Dorothee Herlyn, will provide human combinatorial phage-displayed antibody fragments directed against specific proteins for projects 3, 4, and 5 and pilot projects. To isolate antibody fragments (single chain Fv fragments (ScFv) specifically binding to a selected protein, the staff will use available, highly diverse scFv-phage libraries. ScFv-phage will be subjected to several rounds of panning on antigen-coated plastic surfaces until a plateau of phage enrichment is obtained. ScFv will be expressed in E. coli and purified, followed by extensive characterization of their immunoreactivity, antigen binding specificity and purity. These antibodies will provide the key tools for characterizing tissue distribution of proteins in normal and malignant tissues and for biochemical arid functional analyses, cDNAs, recombinant proteins, and combinatorial antibodies produced under this core will support most projects and pilot studies for current and future investigations on targeting tumor-associated molecules for diagnosis, prognosis and therapy of cancers of the skin.