AfCS > AfCS laboratories > Macrophage Biology Laboratory

ALLIANCE FOR CELLULAR SIGNALING

MACROPHAGE BIOLOGY LABORATORY

San Francisco Veterans Administration Medical Center
University of California, San Francisco
San Francisco, California

Overview

The Alliance for Cellular Signaling (AfCS) Macrophage Biology Laboratory has two major functions. The first is to develop assays and methods for use by the AfCS. As part of this charge, the Laboratory has developed the use of lentiviral-mediated RNAi in RAW264.7 cells, and it is currently testing a panel of cell functions in RAW264.7 cells for possible use as output measurements in the ligand screen.

A second function of the Macrophage Biology Laboratory will be to serve as the primary laboratory for the selective examination of proximal signaling events that modulate Ca2+ flux and/or changes in phosphatidylinositol-3,4,5-trisphosphate (PIP3). This signaling module will serve as a testing ground for larger approaches to cellular signaling with regard to methods, data processing, and data presentation.

Collaborators

The Macrophage Laboratory is collaborating with Cell Signaling Technology (use and development of antibodies) www.cellsignal.com.

Staff

Alliance for Cellular Signaling
Macrophage Biology Laboratory
VAMC 111R 4150 Clement St.
San Francisco, California 94121
Phone: 415-750-2104
Fax: 415-750-6950

Director	William Seaman, M.D.
Associate Director	Eric Brown, M.D.
Lead Scientist	Tamara Roach, Ph.D.
Research Scientist	Robert Rebres, Ph.D.
Research Assistant	David Quan
Research Assistant	Caroline Wong
Research Assistant	Deena Blumenkrantz
Research Assistant	Shuchi Prasad
Lab Administrator	Venecia Jacobs

Slide Presentations from May 23-26, 2004, AfCS Annual Meeting

• "Perturbations/Phenotypes", Bill Seaman
• "RNAi-II", Tamara Roach, Bob Rebres

Goals for Year 5 (9/04-8/05)

• Expand the number of RAW264.7 cells in which targeted reduction in signaling molecules has been achieved through the use of lentiviral vector-mediated RNAi. Include replicate lines to verify phenotypes of interest. Continue population assays of [Ca²⁺]_i flux and phosphoprotein changes.
• With Core E (Molecular Biology Lab), further define the specificity of RNAi in RAW264.7 cells, including testing for off-target effects on both gene and protein expression.
• With Core E, continue to test new lentiviral vectors for efficiency of selection with alternative drug-resistance genes and for efficiency of sorting using membrane expressed protein/epitope markers (e.g., CD4, FLAG, HA). Establish the optimal IRES (IRES1 or IRES2) to provide high and stable expression of resistance genes and markers.
• With Core E, examine gene expression changes in RAW264.7 cells resulting from virus infections, and their transient or stable nature in the lines generated.
• Establish and test RAW264.7 lines with simultaneously by targeted RNAi of 2 and 3 different signaling molecules.
• Explore possible mechanisms for selected phenotypes with additional experimentation; e.g. dose responses to ligands, use of chemical inhibitors, and use of new assays, as directed by the macrophage/FXM committee.
• Supply Core G (Microscopy Lab) with cell lines in which RNAi has been achieved without the use of fluorescent markers to facilitate studies of PH-domain at the single-cell level. Continue to supply all lines to Core G for single cell [Ca²⁺]_i flux assays.