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TRAMP
Q: Where can I find a brief overview of the TRAMP model? A: We have a short perspective available in PDF CLICK HERE A paper describing the pathobiology of prostate cancer progression in TRAMP can be downloaded here ----- TRAMP Breeding Q: Currently we are breeding the TRAMP mice. From your website, you recommend to breed heterozygous (TRAMP+/-) females with non-transgenic C57BL/6 breeder males. Now I have one question, if the TRAMP male mice produced from homozygous TRAMP female mated with homozygous TRAMP male, will the tumors in those mice be more malignant or tumor onset accelerate? A: We do not usually do that because the site of transgene integration in fact represents an insertional event and while chances are slim that the transgene inactivates an essential gene, double (homozygous) transgenix would have the increased possibility of being "double knockouts" at this site (this would severely complicate analysis). single allele transgenix are at worse heterozygous at the site of integration meaning the "sister" chromosome would still (probably) be wildtype and fully functional. Our recommended breeding scheme is available in PDF CLICK HERE ----- PB-Tag Construct Q: Where can I find a map of the PB-Tag construct used to generate TRAMP? A: A complete description is available in PDF CLICKHERE ----- Q: Where can I find a PCR based screening protocol for the TRAMP mice? A: A complete screening protocol is available n PDF CLICK HERE ----- Q: Is the TRAMP transgene integrated into the genome of the mouse? If so, are the locations and copy number known? A: The PB-Tag transgene is integrated into the genome. We do not know the exact location or copy number at this time. ----- TRAMP and Pre-clinical Trials Q: I want to test a discrete compound in TRAMP. Do you have a recommended protocol? A: A guideline for pre-clinical trials involving the TRAMP mode is available. CLICK HERE ----- SV40 and TRAMP Tissues A: I am sorry to learn of this accident. The mice did not to my knowledge at time of shipping contain any known live SV40 virus. The PB-Tag transgene, which is stably integrated into the genome carries only the minimal probasin promoter (-426/+28) driving the early region (encoding large T and small t open reading frames). The construct would preferentially express in prostatic tissue. No other SV40 viral genes are known to be present and no infectious SV40 should be produced as a consequence of our engineering. It is unlikely this mishap would expose you in any way to any infectious material related to the PB-Tag transgene. However, your mice were not under my care so I can not attest to their health status or likelihood of their exposure to other infectious material(s) after leaving my lab. Due diligence warrants that you should take all precautions when working with live material. I would consult the veterinarian responsible for their health at your institute and obtain the most recent serologic report from sentinel animals housed in the animal room that housed the mouse that was the source of tissues you were using. ----- VX-680 Q: I saw your paper describing the use of Aurora Kinase Inhibitor VX-680. Where can I order this inhibitor? A: Here is all the information we have for KAVA Technologies: We have recently found VX-680 (and other Aurora inhibitors) can be obtained from American Custom Chemicals Corp. For further information please click here or visit their website: http://www.acccorporation.com Also see Nature Medicine 13 p. 511(2007) ----- TRAMP-C Cell Lines and Transfection Q: C-1 and C-2 cell lines are not neo-resistant, correct? Have you managed to stably transfect these cells? A: These cells do not carry a construct with a "neo resistant" allele. However, they do seem to show a naturally high tolerance for G418. We have transiently transfected them. We do not know if they can be stably transfected. ----- Magnafire SP Cameras and Mac OSX Q: I have a Magnafire SP digital firewire camera from Olympus and the accompanying Magnafire 2.0 software from Optronics is no longer compatable with Mac OS X 10.4.9. The software is proprietary as is the camera. What can I do? The software may launch once then fail to recognize the FireWire camera. Optronics claims they have no plans to update the software. Is there a workaround or patch? A: Yes. We have devised a pretty good workaround for this problem. You can download the PDF by cclicking HERE ----- TRAMP and Immune Tolerance Q: Our group is studying immune tolerance to Tag in the TRAMP mouse, our PCR and qRT-PCR show expression in the prostate but do not show any expression of Tag in the thymus, which is consistent with your published data and data from other groups who use the same mouse model. However, there is a report in the Journal of Immunology from 2002 (please see below) that contradicts this finding. How I can show that this paper is wrong or right? Zheng X, Gao JX, Zhang H, Geiger TL, Liu Y, Zheng P. Clonal deletion of simian virus 40 large T antigen-specific T cells in the transgenic adenocarcinoma of mouse prostate mice: an important role for clonal deletion in shaping the repertoire of T cells specific for antigens overexpressed in solid tumors. J Immunol. 2002 Nov 1;169(9):4761-9. A: Some say that SV40 T and t are expressed in the young thymus - others think not. Clearly you have looked and find no expression by sensitive assays. While we accept that most genetically engineered mouse models are tolerant of their transgenic payloads, there is precedence (mostly from Doug Hanahan's RIP-Tag mice in the 80's) that the expressivity of the construct (timing and tissue distribution) can influence the degree of tolerance. I have long suspected that a careful quantitative examination of the expression of the T and t transcripts in the developing, juvenile, young adult and aged thymus would be a good study. Recently, I received a piece of unpublished data from Dr. Arthur Hurwitz showing that indeed Tag is differentially expressed in thymus as a function of development. The expression of the SV40 T antigen transgene was detected by RT-PCR and Southern Blotting. The RNA was isolated from Bladder, Lung, Thymus and Prostate at various times after birth. Clearly expression of the Tag transcript is highest in thymus at 7.5 weeks corresponding to puberty in the males. The expression in adults at 15 and 23 weeks is significantly reduced. Dr. A. Hurwitz is at NCI-FCRF <hurwitza@ncifcrf.gov>
----- Q: Some months ago you kindly sent us TRAMP-C1 and -C2 cells for our work. These cells grow perfectly in vitro. Nevertheless, when injecting the cells subcutaneously into the hindquarter of 13 weeks old male C57Bl/6J mice no tumors develop. Even after resolving and injectiong cells in matrigel solution no tumors developed. The matrigel solution is commercially available and is a solubulized basement membrane preparation extracted from EHS mouse sarcoma. We don't know, how to solve this problem. Does matrigel hinder tumor grwoth (e.g. in a somehow allergic reaction?) Are the animals too youg or too old? ----- TRAMP and Androgen Signaling Q: Recently I submitted a NIH grant proposing to study a putative oncogene by crossing TRAMP mouse to a mouse with an oncogene mutation. One of the critiques was "if knockout of the oncogene potentially affects AR, how do you know changes in tumor incidence in the hybrid mouse is resulted from the elimination of the oncogene or from modifying the PB promoter activity by AR because the PB promoter contains AR binding sites." How do I address this issue? A: As with any model system there are limitations and you need to appreciate these when designing your studies. Since the SV40 T/t sequence is under transcriptional control of the rat Probasin (PB) promoter and PB is, in part regulated by androgen action, then you would need to consider the possibility that any change that can influence the level of PB promoter activity could change the level of SV40 T/t expression. This would be true of any "model" system that relies on enforced expression / repression of any gene. We usually try to measure levels of SV40 T in a compound model by western blot or IHC to control for this possibility. See Figure 4D in the publication by Chung et al., Cancer Res 2007 67(12):5965-75. |
