tissue culture dish vs growth area

pc12 information

Cell Biology

ATCC® Number: CRL-1721™ Price: $256.00
Additional information about this cell line
Designations: PC-12 Depositors: B Patterson
Biosafety Level: 1 Shipped: frozen
Medium & Serum: See Propagation Growth Properties: loosely adherent, multicell aggregates
Organism: Rattus norvegicus (rat) Morphology: polygonal


Source: Organ: adrenal gland
Disease: pheochromocytoma
Cellular Products: catecholamines; dopamine; norepinephrine [1163]
Permits/Forms: In addition to the MTA mentioned above, other ATCC and/or regulatory permits may be required for the transfer of this ATCC material. Anyone purchasing ATCC material is ultimately responsible for obtaining the permits. Please click here for information regarding the specific requirements for shipment to your location.

Related Cell Culture Products
Applications: transfection host (Roche FuGENE® Transfection Reagents
technology from amaxa)
Receptors: nerve growth factor (NGF), expressed
Tumorigenic: YES
Cytogenetic Analysis: 40 chromosomes; 38 autosomes plus XY [1163]
Gender: male
Comments: The PC-12 cell line was derived from a transplantable rat pheochromocytoma. [1163]
The cells respond reversibly to NGF by induction of the neuronal phenotype. [1163]
The cells do not synthesize epinephrine. [1163]

Propagation: ATCC complete growth medium: The base medium for this cell line is ATCC-formulated F-12K Medium, Catalog No. 30-2004. To make the complete growth medium, add the following components to the base medium: fetal bovine serum to a final concentration of 2.5%; horse serum to a final concentration of 15%.

Atmosphere: air, 95%; carbon dioxide (CO2), 5%
Temperature: 37.0°C

Subculturing: Protocol: Volumes used for this protocol are for a 75cm2 flask; proportionally reduce or increase amount of dissociation medium for culture vessels of other sizes.1. Remove and discard old culture medium.2. Pipet 10 ml fresh medium over the cell sheet and scrape.3. Aspirate cells with a small bore pipette to break up clusters.4. Add appropriate aliquots of the cell suspension to new 75 cm2 flask with 15 ml fresh growth medium. Seed flask at 1.0 x 10(4) to 3.0x 10(4) viable cells / cm2.Or use subcultivation ratio of 1:3 twice weekly Subculture when cell density reaches between 1.0x 1 0(5) to 2.0x 10(5) viable cells / cm2.5. Place culture vessels in incubator at 37ºC.PC-12 cells adhere poorly to plastic and tend to grow in small patches of loosely attached cells.Attachment can be enhanced by coating the flasks with Bovine Collagen I or using Corning® CellBIND® Surface Flasks (Free Samples)

Subcultivation Ratio: 1:3 twice weekly
Medium Renewal: Every 2 to 3 days
Preservation: Freeze medium: Complete growth medium supplemented with 5% (v/v) DMSO

Storage temperature: liquid nitrogen vapor phase
Doubling Time: 48 hrs
Related Products: Recommended medium (without the additional supplements or serum described under ATCC Medium):ATCC 30-2004
recommended serum:ATCC 30-2020
recommended serum:ATCC 30-2040
References: 1162: Levi A, et al. Molecular cloning of a gene sequence regulated by nerve growth factor. Science 229: 393-395, 1985. PubMed: 3839317
1163: Greene LA, Tischler AS. Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor. Proc. Natl. Acad. Sci. USA 73: 2424-2428, 1976. PubMed: 1065897
22344: Biocca S, et al. A macromolecular structure favouring microtubule assembly in NGF- differentiated pheochromocytoma cells (PC12). EMBO J. 2: 643-648, 1983. PubMed: 6641712
33014: Weber E, et al. Distinct functional properties of Rab3A and Rab3B in PC12 neuroendocrine cells. J. Biol. Chem. 271: 6963-6971, 1996. PubMed: 8636125

things to do

1.dissertation writing
2.inject col in mouse col for behavioral study
inject LPS/MPP/COL in mouse striatum for TNF measurement
3.find out whether rab11 is invovled in the secretion of TNF in microglia
4.find out whether lentivirus can activate microglia
5.discuss with Yin about the interference of OCM in striatum of rat

Andrew Dillin

Andrew Dillin, Pioneer Developmental Chair and an associate professor in the Molecular and Cell Biology Laboratory, uses the tiny roundworm Ceanorhabditis elegans to study the process of aging by looking at a hormone that is most widely recognized for its role in diabetes among humans: insulin. The insulin signaling pathway in worms is not only almost identical to that found in humans, but Dillin discovered that insulin also controls many physiological aspects in the worm's body, including reproduction and aging. In humans, interfering with insulin/IGF-1 signaling to generate a life-prolonging benefit would lead to type 2 diabetes and possibly cancer. In worms, larval development and reproduction are affected along with longevity.


Some of Dillin's earlier research had hinted at the possibility to genetically manipulate one element of the pathway without disrupting its additional functions, this led him to search for "specificity" factors that may control how and if insulin and IGF-1 impact a wide range of target genes. Recently, he and his team pinpointed a protein specifically responsible for extending lifespan and youthfulness without disrupting the worms' response to some forms of stress, development and fertility controlled by the insulin signaling pathway.


Additionally, Dillin is interested in age-onset neurodegenerative diseases. Like most neurodegenerative diseases, Alzheimer's disease usually appears late in life, raising the question of whether it is a direct and disastrous consequence of aging or if the toxic protein aggregates that cause the disease simply take a long time to form. He discovered that the harmful beta amyloid aggregates accumulate when aging impedes two molecular clean-up crews from getting rid of these toxic species.

Education
B.S., Biochemistry, University of Nevada, Reno
Ph.D., Molecular & Cell Biology, University of California, Berkeley
Awards and Honors
McKnight Neurosciences of Brain Disorders Award (2007)
Ellison Medical Foundation Award (2004-2008)
Larry L. Hillblom Junior Faculty Award (2003-2006)
American Diabetes Association Junior Faculty Award (2004-2006)
Damon Runyon-Walter Winchell Postdoctoral Fellowship, UC San Francisco (1999-2002)
Selected Publications
Cohen E, Bieschke J, Perciavalle RM, Kelly JW, Dillin A. Opposing Activities Protect Against Age Onset Proteotoxicity. Science. 2006.
Wolff S, Ma H, Burch D, Maciel GA, Hunter T, Dillin A. SMK-1, an essential regulator of DAF-16-mediated longevity. Cell. 124(5):1039-53; 2006.
Raices M, Maruyama H, Dillin A, Karlseder J. Uncoupling of longevity and telomere length in C. elegans. PLoS Genet. (3):30; 2005.
Dillin, A., Hsu, A., Arantes-Oliveira, N., Lehrer-Graiwer, J., Hsin, H., Fraser, A. G., Kamath, R. V., Ahringer, J., Kenyon, C. Lifelong Rates of Behavior and Aging Specified by Respiratory Chain Activity During Development. Science. 298(5602):2398-401; 2002.
Dillin, A., Crawford, D. K., Kenyon C. Timing requirements for insulin signaling in C. elegans. Science. 298:830-4; 2002.
Arantes-Oliveira N, Apfeld J, Dillin A, Kenyon C. Regulation of life-span by germ-line stem cells in C. elegans. Science. 295(5554):502-5; 2002.

Michael G Rosenfeld

http://rosenfeldlab.ucsd.edu/
We are investigating the molecular strategies used to generate genome-wide, integrated transcriptional responses to the vast signaling network that regulates development and homeostasis. We are using genetic, biochemical and biological approaches to define the strategies used by the human body to specify modifications to development and differentiation of cells when genes are expressed. Our work has revealed unexpected gene-specific strategies that link regulated gene responses to other cellular response programs, including DNA damage and DNA repair. Defining these strategies has suggested new approaches to diseases, including growth defects, diabetes, arteriosclerosis, and several prevalent forms of cancer.

Regulatory Strategies in Development and Function of the Neuroendocrine System

Molecular Mechanisms of Regulated Gene Transcription

Neural Stem Cells--Corepressor Strategies in Neuronal Development

Macrophage: Cancer Cell Interactions in Breast and Prostate Cancers

Sensors and Signals--coactivator and corepressor complexes in genome-wide patterns of gene expression

Neurodegenerative Diseases

Tom A Rapoport

Department of Cell Biology
Harvard Medical School/HHMI
LHRRB Building, Room 613
240 Longwood Avenue, Boston, MA 02115-6091

tel: (617) 432-0637; fax: (617) 432-1190
email: tom_rapoport@hms.harvard.edu

Research Interests:
We are interested in the molecular mechanisms by which proteins are transported across the eukaryotic endoplasmic reticulum (ER) membrane or across the bacterial plasma membrane. Proteins are transported through a protein-conducting channel, formed from a conserved trimeric membrane protein complex, called the Sec61p complex in eukaryotes, and the SecY complex in prokaryotes. The channel is a passive pore that needs to associate with partners that provide the driving force for translocation. We know of three different pathways in which the channel functions.

The first is co-translational translocation, in which the ribosome is the major partner. Our present work concentrates on the structure of ribosome-channel complexes using electron cryo-microscopy single particle analysis (collaboration with the group of Christopher Akey at Boston University) and X-ray analysis.

The second mode is post-translational translocation in eukaryotes, in which another membrane protein complex, the Sec62/63p complex, and the luminal BiP protein are the partners.

The third mode is posttranslational translocation in bacteria, in which the ATPase SecA is the major partner. Our goal is to elucidate the mechanism by which the ATPase SecA provides the driving force for translocation.

A major effort in the lab is directed towards high-resolution structures of the translocation channel. We have obtained X-ray structures of the SecY complex from an archaebacterium, and more recently, of a SecA-channel complex. Together with biochemical data, these results suggest mechanisms for how the signal sequence of a substrate is recognized, how polypeptide chains are moved through the channel, and how trans-membrane segments of membrane proteins are integrated into the lipid bilayer.

We are also interested in the process by which misfolded ER proteins are transported back into the cytosol for degradation by the proteasome, a process called ERAD (for ER-associated protein degradation) or retro-translocation. We have provided evidence that there exist three ERAD pathways in yeast, depending on where the misfolded domain of the ER protein is located (ERAD-L for proteins with misfolded luminal domains, ERAD-M for proteins with misfolded intra-membrane domains, and ERAD-C for membrane proteins with misfolded cytosolic domains). The pathways use different ubiquitin-ligase complexes but converge at the Cdc48p ATPase complex (p97 or VCP complex in mammals). We are currently using translocation intermediates and crosslinking methods to identify the retro-translocation channel that likely exists at least for ERAD-L substrates.

A major effort in the lab concerns the mechanism by which the structure of the ER is generated and maintained. Our results show that the reticulons and DP1/Yop1p shape the tubular ER. We are interested in understanding how these proteins are regulated and how ER sheets are generated.

Other Projects Include:
1. The mechanism by which DNA is transported across membranes during the sporulation of B. subtilis.
2. The structure a bacterial homolog of the VKOR protein (vitamin K epoxide oxidoreductase).
3. The mechanism by which mRNA is transported in mammalian cells.



Selected Publications:
van den Berg, L., Clemons, W., Collinson, I., Hartmann, E., Modis, Y., Harrison, S.C., and Rapoport, T.A. (2004). X-ray structure of a protein-conducting channel. Nature 427: 36-44.

Carvalho, P., Goder, V., and Rapoport T.A. (2006). Distinct ubiquitin-ligase complexes define convergent pathways for the degradation of ER proteins. Cell 126: 361-373.

Voeltz, G.K., Prinz, W.A., Shibata, Y., Rist, J.M., and Rapoport, T.A. (2006). A class of membrane proteins shaping the tubular endoplasmic reticulum. Cell 124: 573-86.

Rapoport, T.A. (2007). Protein translocation across the eukaryotic ER and bacterial plasma membranes. Nature 450: 663-669.

Zimmer, J., Nam, Y., and Rapoport, T.A. (2008). Structure of a complex of the ATPase SecA protein-translocation channel. Nature 455(7215): 936-43.

Susan Ferro-Novick

Professor of Cell Biology
Investigator, Howard Hughes Medical Institute
Phone: (203) 737-5207
Lab: (203) 737-4453/-4451
Fax: (203) 737-5246
e-mail: susan.ferronovick@yale.edu Department of Cell Biology
Yale Universtiy School of Medicine
333 Cedar Street
PO Box 208002
New Haven, CT 06520-8002
Vesicle traffic and organelle inheritance
The goal of our research program is to understand how the specificity of vesicle traffic is maintained and how organelles are inherited from mother to daughter cells.

Vesicle Traffic
For our studies on vesicle traffic, we have focused on the multiprotein complex called TRAPP. There are two forms of the TRAPP complex, TRAPP I and TRAPP II. TRAPP I is required for membrane traffic from the endoplasmic reticulum (ER) to the Golgi, while TRAPP II is required for traffic from the early endosome to the Golgi and within the Golgi. Interestingly, spondyloepiphyseal dysplasia tardia, a recessive disorder in bone formation is caused by mutations in the human orthologue of a TRAPP subunit.

Using a vesicle binding assay that employs in vitro formed ER-derived vesicles and pure TRAPP I, we have demonstrated that TRAPP I specifically binds to ER to Golgi vesicles. These findings imply that TRAPP I plays a key role in conferring the specificity of ER to Golgi vesicle traffic. TRAPP I binds to coated ER-derived vesicle via an interaction with the coat cargo adapter complex (see Figure), linking TRAPP I dependent vesicle binding to cargo recognition. Once binding occurs, TRAPP I activates the small GTPase Ypt1p, converting it from its GDP-bound to its GTP-bound form. The activation of Ypt1p by TRAPP I may be the signal that the vesicle has reached its correct acceptor compartment. This then leads to the recruitment of other components, such as Uso1p. The pairing of the SNAREs, a class of membrane proteins that are required for membrane fusion, is the final step in docking an ER-derived vesicle to the Golgi.

Organelle Inheritance
It is the goal of these studies to define the process by which the ER is delivered into daughter cells. To achieve this goal a genetic approach has been used to identify the machinery that moves ER tubules from mother to daughter cells. This approach has led to the identification of a collection of genes whose products are required for ER inheritance. A track and motor that moves ER tubules into daughter cells, as well as a putative receptor for cortical ER in daughter cells have been identified. Orthologues of these components are present in higher cells.


Selected Publications
Cai, H., Reinisch, K. and Ferro-Novick, S. 2007. Coats, Tethers , Rabs and SNAREs work together to mediate the intracellular destination of a transport vesicle. Dev Cell 12: 671-682.

Cai, H., Yu, S., Menon, S., Cai, Y., Lazarova, D., Fu, C., Reinisch, K., Hay, J. C. and Ferro-Novick, S. 2007. TRAPPI tethers COPII vesicles by binding the coat subunit Sec23p. Nature 445: 941-944.
(Commentaries on this paper appeared in Dev Cell and Curr Biol)

Du, Y., Walker, L., Novick, P. and Ferro-Novick, S. 2006. Ptc1p regulates cortical ER inheritance via Slt2p. EMBO J. 25: 4413-4422.
(This paper was highlighted in EMBO J)

Yu, S., Satoh, A., Pypaert, M., Muller, K., Hay, J.C. and Ferro-Novick, S. 2006. mBet3p is required for homotypic COPII vesicle tethering in mammalian cells. J. Cell Biol. 174:359-368.

DeCreane, J.O., Coleman, J., Estrada de Martin, P., Pypaert,M., Anderson, S., Yates III, J.R., Ferro-Novick, S and Novick, P. 2006. Rtn1p is involved in structuring the cortical ER. Mol. Biol. Cell 17:3009-3020.

Cai, H., Zhang, Y., Pypaert, M., Walker, L. and Ferro-Novick, S. 2005. Trs120p mediates traffic from the early endosome to a late Golgi compartment. J. Cell Biol. 171:823-833.

Estrada de Martin,P., Novick,P. and Ferro-Novick,S. 2005. The organization, structure and inheritance of the ER in higher and lower eukaryotes. Biochem. Cell Biol. 83:752-761.