AfCS > AfCS laboratories > Molecular Biology Laboratory

ALLIANCE FOR CELLULAR SIGNALING

MOLECULAR BIOLOGY LABORATORY

California Institute of Technology
Pasadena, California

Overview

The Alliance for Cellular Signaling (AfCS) Molecular Biology Laboratory is involved in four major areas: transcript analysis, nucleic acid-based inhibition of gene expression, cDNA construction, and yeast 2-hybrid screens.

The lab is currently utilizing several microarray array platforms, and they have been used to assess transcript profiles in primary B cells, B cell lines, myocytes and macrophages stimulated with a variety of ligands for a wide range of receptors. The lab has constructed inkjet-printed 16K cDNA arrays in collaboration with Agilent Technologies using the RIKEN FANTOM library. Since the ideal mouse microarray is evolving continually, the lab has also constructed 16K oligonucleotide-based arrays. Continuing microarray development will allow for the complete representation of genes from the AfCS protein list and the flexibility to develop a whole mouse genome chip in the not too distant future. Microarrays are currently being used to assess transcript profiles in the RAW264.7 mouse macrophage cell line. For data analysis, the group has off-line computer workstations equipped with the latest expression analysis software.

The Molecular Biology Laboratory is using RNA interference to inhibit the expression of target genes in RAW264.7 cells. We are generating both siRNAs (designed by Dharmacon’s SMARTpool algorithm) and shRNAs against our target genes. This permits both short-term transient knockdown (siRNAs) and also longer term studies through the creation of stable RAW cell lines using lentivirally expressed shRNAs (Macrophage lab).

A further role of the Molecular Biology Laboratory is to construct and provide plasmid constructs for use in other AfCS laboratories. The lab has established the use of the Gateway cloning system (Invitrogen Life Technologies), which facilitates the transfer of cloned cDNAs or shRNAs into multiple expression systems in a single step using the site-specific recombination sites of lambda phage. The lab has constructed vectors that allow Gateway cloned cDNAs to be expressed in many different expression systems with a variety of fusions such as GFP variants or epitope tags. Similarly, lentiviral vectors have been developed for shRNAs expression containing a variety of markers and drug selection genes to permit the creation of shRNA-expressing stable cell lines. Over one thousand cDNAs/shRNAs have been cloned thus far, and they have been used to generate over five thousand expression constructs. Almost three thousand of these constructs have been distributed to the ATCC, and construct details are available in our plasmid database at http://www.signaling-gateway.org/data/plasmid/.

Other projects in the Molecular Biology Laboratory involve collaboration with industrial partners. In association with Isis Pharmaceuticals, Inc., the lab is using antisense oligonucleotides to complement the RNAi studies described above. In collaboration with Myriad Genetics, Inc., the lab carries out yeast 2-hybrid screens at a rate of 500 baits per year (this will permit the analysis of approximately 100 selected genes per year). The yeast 2-hybrid project has identified over 1000 protein-protein interactions, all of which are available at the following website (http://www.signaling-gateway.org/data/Y2H/cgi-bin/y2h.cgi).

Collaborators

The Molecular Biology Laboratory is collaborating with Isis Pharmaceuticals, Inc., (use of antisense oligonucleotides), Myriad Genetics, Inc., (performance of yeast two-hybrid screens), Dharmacon, Inc., (SMARTpool siRNAs) and Cell Signaling Technology (use and development of antibodies) www.cellsignal.com.

Staff

Alliance for Cellular Signaling
Molecular Biology Laboratory
California Institute of Technology
Division of Biology 147-75
1200 E. California Blvd.
Pasadena, California 91125
Phone: 626-395-3789
Fax: 626-796-7066

Director	Melvin Simon, Ph.D.
Lead Scientist	Iain Fraser, Ph.D.
Lead Scientist	Sangdun Choi, Ph.D.
Research Scientist	Pamela Eversole-Cire, Ph.D.
Data Analyst	Xiaocui Zhu, Ph.D.
Research Assistant	Mi Sook Chang
Research Assistant	Christine Kivork
Research Assistant	Jamie Liu
Research Assistant	Ji Young Park
Research Assistant	Leah Santat
Research Assistant	Estelle Wall
Research Assistant	Joelle Zavzavadjian
Research Assistant	Jacqueline Zhou
Lab Administrator	Josephine Macenka

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

• "Transcripts",   Mel Simon
• "Y2H Database; Plasmid Database, Reagents & ATCC",   Iain Fraser
• "RNAi-I",   Iain Fraser

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

1. Transcript analysis
   • Use the Agilent inkjet printed Operon + Compugen 16K oligo arrays for selected single and double ligand screens in RAW 264.7 cells.
   • Select additional ligand combinations based on emerging questions from other single and double screen experiments such as Bio-Plex cytokine array.
   • Perform transcriptional analyses of wild type RAW cells vs. cells perturbed by various RNAi approaches.
   • Include Affymetrix GeneChip analyses with extended gene sets for special experiments.
   • Perform Bio-Rad iCycler iQ 4-Color Real Time Detection PCR to routinely provide quantitative measurement of selected gene targets for ongoing projects in a high-throughput mode.
2. Inhibition of gene expression
   • Continue RNAi reagent development program to validate further siRNAs and shRNAs for use in AfCS studies
   • Acquire Hannon lab shRNA library and assess as a potential source of effective RNAi reagents
   • Investigate utility of shRNA library for large-scale perturbation screens in RAW cells
   • Continue to develop vector systems to facilitate application of RNAi technology in RAW cells
   • Continue use of existing antisense reagents as a complement to RNAi in RAW cell signaling assays
3. cDNA cloning
   • Continue with cloning and sequencing of high priority FXM full-length cDNAs
   • Implement further cDNA cloning and vector development projects on an 'as needed' basis for AfCS studies.
   • Streamline process for plasmid distribution through the ATCC.
4. Protein-protein interactions
   • Coordinate the selection of further signaling proteins for yeast 2-hybrid screens in collaboration with Myriad Genetics.
   • Use yeast 2-hybrid interactions from FXM baits to expand parts list and facilitate interpretation of FXM data.