- How can I obtain DNA for the RCAS vectors and adaptors?
If you are at a nonprofit research laboratory and have no commercial interest in the RCAS system, we can help you execute an NIH simple letter agreement (SLA). Once the SLA forms are completed, we will ship the plasmid DNA to you. If you are at a commercial organizations or have any commercial interests, you must negotiate directly with the Technology Transfer Center, National Cancer Institute.
- How can I obtain DF-1 cells?
The permanent EV-0-derived chicken cell line DF-1 was prepared by Doug Foster (University of Minnesota) and can be obtained from the American Type Culture Collection (ATCC; catalog #CRL-12203). Note that this cell line is not listed in the ATCC website, but is available through telephone orders (800-638-6597 or 703-365-2700).
- How can I obtain transgenic mice that express tva?
This requires both an SLA (see FAQ #1) and official assurances for animal welfare. Shipping the animals can be expensive (particularly to Europe). We can help with the paperwork; anyone with a commercial interest will have to negotiate with the TDCB, NCI.
- Where can I get the sequence of the vector?
We have provisional sequences (downloadable as text files) for several of the commonly used RCAS vectors: RCASBP(A), RCASBP(B), and RCASBP(M). Numbering of these sequences begins at the U3 R
boundary in the lefthand LTR. Note that the sequences have not been finalized. We are in the process of preparing the final versions of the sequences. As soon as this has been done, the corrected sequences will be posted here.
- If the ClaI cloning site is unique, why does the sequence have two ClaI sites listed?
As strange as it must seem now, the original RCAS cleavage site map was determined by digestion with restriction enzymes, not from the sequence. The upstream ClaI site is subject to Dam methylation in E. coli, and is not cleaved if the plasmid DNA is grown in a Dam(+) E. coli strain.
- What RCAS vectors are available?
This website should give you a reasonable idea of the vectors we can supply. If you want something exotic, we probably can help you find what you need, or give you the materials you need to construct the vector you want.
- I’m having trouble putting inserts into the ClaI site — What’s the problem?
Either bad DNA or bad ClaI. There doesn’t seem to be any restriction on putting inserts into the ClaI site in the RCAS plasmids and cloning the plasmid in E. coli. ClaI is not the most reliable restriction enzyme; we’ve been sold bad batches. If the digested DNA looks fine on a gel but won’t ligate, the ClaI probably contains some 3' DNA exonuclease, a problem that was much worse 5–10 years ago. If you are having such problems, check your ligation reaction before you do the transformation.
- I don’t get any virus back after the cells are transfected — What’s wrong?
Basically, if you use a simple transfection protocol to put 5-10 micrograms of RCAS DNA onto a plate of compatible chicken cells, you will get virus back. If you are using CEFs, make sure the cells have the receptor for the envelope you have chosen. Also, make sure your cells are not contaminated with an ASLV that will block the replication of the RCAS vector.
- I get back replicating virus, but the insert is gone — What’s wrong?
Unfortunately, there are a number of possibilities; refer to Overview — What to Avoid in Vector Design.
- My institutional biosafety committee (IBC) wants to know how I plan to handle the vectors — What do you do?
If you use the RCAS vectors with avian envelopes, the viruses will not infect mammalian cells. Even the versions that have murine envelopes, which can infect mammalian cells, do not replicate in mammalian cells. That having been said, we recommend that the vectors be handled carefully because they are infectious agents. We handle the viruses with avian envelopes under BL-1 condition and those with mammalian envelopes under BL-2.