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bacTRAP project










About Translating Ribosome Affinity Purification (TRAP):

The mammalian central nervous system (CNS) is a highly sophisticated network of hundreds of cell types and sub-types, each with unique functions and corresponding molecular profiles. Often, diseases of the CNS are the consequence of perturbations of a single cell type, yet many cell types remain only vaguely described. We developed the BacTRAP methodology which allows us to isolate the complete suite of translated cell type specific mRNAs directly from transgenic animals [1]. Bacterial Artificial Chromosomes (BACs) were used to drive cell type specific expression of ribosomal proteins fused with eGFP. Tagged ribosomes are readily affinity purified along with the suite of cell-specific translating mRNAs. BacTRAP translational profiling permits comprehensive studies of translated mRNAs in genetically defined cell populations, and their responses to physiological perturbations. Our initial survey of the mouse brain covered 24 cell populations including a variety of neurons and glia [2], demonstrating the wide utility of the method.

About www.bactrap.org:

BacTRAP.org currently provides a location for Downloads of protocols for TRAP, links to publications and data, and R code for useful modules analysis of bacTRAP microarray data. It also provides information about FAQs, Contacts for mouse, antibody, and plasmid, requests, information on the Users Group, the people behind bacTRAP.org, and the Funding that makes this all possible.

In the near future, BacTRAP.org will also provide three methods to query our data: First, the ability to query at a glance the expression and specificity [3] of your favorite gene across all of our initial bacTRAP survey of cell types, second, the ability to filter for large sets of genes specific to or expressed in your favorite cell type, and finally the ability to visualize the anatomical expression of the TRAP transgene across all of our available lines of mice to determine if one of these lines may target an additional cell type you are interested in, beyond our initial survey.

FAQs:

As this site has just been launched, there are no questions yet that could be said to have been asked frequently...

Contacts:

This website was created by Joe Dougherty, Nick Didkovsky, and Juliet Zhang. Database design and Java/JSP programming by Nick Didkovsky. It is built from data generated by many individuals [1,2,3], and with image processing assistance from John Baker.

Contact information for members of the Heintz lab is found here.

Users Group:

For those interested in using the TRAP technique, a Google groups has been started to share hints and methodology for Translating Ribosome Affinity Purification (TRAP), and the bacTRAP variation.
http://groups.google.com/group/trapists/

Sources of Funding Include:

Howard Hughes Medical Institute, the Adelson Program in Neural Rehabilitation and Repair, the Simons Foundation, the Conte Center (NIH/NIMH 5P50MH074866 P2), and the Croll Charitable Trust.

Materials Requests:

For requests of bacTRAP mice: heintz [at] rockefeller.edu
A simple MTA will be required. Currently most lines are privately banked as sperm with JAX.

For requests of plasmids containing TRAP construct: walshj [at] rockefeller.edu
A simple MTA will be required. Providing a Fedex shipping number is considered polite.

For request of monoclonal eGFP antibodies (from MSKCC core): weisgarf [at] mskcc.org

It is recommended to request two monoclonals, clones Htz-GFP19C8 and Htz-GFP19F7, 50 ugs of each per experiment. For TRAP, we have been using a mixture of both, in the form of bioreactor supernatant. No MTA required. Follow the procedure below.

  1. To register at MSKCC website ( http://macfwebext.mskcc.org/ ) as "Other User"
    • You will need to provide your...
    • name / phone number and email address
    • shipping address
    • billing address
      1. This is almost always on packing slips - Your purchasing should be able to provide this - usually has "Accounts Payable" in it

  2. To decide what form you want for the MAb? - MSKCC distributes:
    • Bioreactor supernatant (BRS) - conditioned media containing... 1 mg MAb/ml on average - base media - 0.5% FBS - cell debris
    • Purified MAb (in PBS / no azide)
    • Conjugated Mabs (if we are not over booked)

  3. To know how much you want? - A pricing spreadsheet is available on the website after login – but here are some general guidelines:
    • The smallest aliquot of purified MAb is 1 mg
    • The smallest aliquot of BRS varies - but is no bigger than 10 mg/tube
    • Purified tends to run 4 to 5x higher than BRS
    • The more you purchase the less it is per mg
    • After 50 mg the differences per mg are minimal
    • For TRAP, figure 50 ugs of each antibody per experiment

  4. Once you have a PO# you can submit the order online:
    • Go to the left hand navigation bar and under "established MAbs" choose "Shop for a MAb"
    • Select the appropriate "HtzGFP-" MAb from the list and hit search
    • Complete the rest of the forms

  5. FedEx (or other overnight carrier) account number - put in the comments section of the order:
    • Shipping is NOT built into the cost of the MAb - so MSKCC will have the carrier bill you directly

  6. We will ship it out on a Monday/Tuesday/Wednesday:
    • MSKCC will email you the tracking # so you know if there is a delay
    • MSKCC will track it ourselves too - so we have confirmation of delivery - for our billing purposes

Questions? Contact Dr. Weis-Garcia (weisgarf [at] mskcc.org)

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