Transgenic
and
In Vivo Mutagenesis
Interest Group
Newsletter, April 26, 2001
Environmental Mutagen Society Special Interest Group in: Transgenic and In Vivo Mutagenesis
This is a report on the meeting of the Transgenics Special Interest Group, March 18, 2001 at the EMS meeting in San Diego.
Standard Disclaimer : This Newsletter does not necessarily reflect the opinions or positions of the EMS or its members. Any errors or omissions are strictly on the part of the secretary.
Business Stuff :
(not arranged exactly in order of occurrence)
Meeting was brought to order by Carrie Valentine, chair. Attendance was recorded by a hand-out email list : 35 people recorded their email address. Nominations and elections for chair and secretary were held. By consensus, Carrie Valentine [cvalentine@nctr.fda.gov ] will remain as chair, and Barry Ford [barry.ford@drdc-rddc.gc.ca]agreed to continue as secretary for the coming year.
Due to the success of the group, encouragement from the Professional Development and Membership Committee, and leftover from last year (as in not done), Barry Ford will develop webpages and links relevant to TIVM for the EMS website. It is hoped that this page will serve as a template for other groups in EMS. Anyone who has URLs (annotated please) which may be of interest should forward these to BF. Also, BF is looking for materials which might be contributed to the public domain via the website, such as overview slides, good slides explaining methodological aspects, or summary data. Image formats such as tiff, gif, bmp, Powerpoint, Harvard Graphics, Freelance, Corel, etc can be used. Don’t be shy. Send them directly to the editor [barry.ford@drdc-rddc.gc.ca].
Science Stuff :
Heinrich Malling [malling@niehs.nih.gov] discussed the phiX model. Current work has improved the ligation system, allowing about 10% of extracted phage DNA to be packaged. He reports competitive inhibition with host DNA, however, recovery of phiX using the present methods is more than sufficient for mutation detection. The original PhiX assay detects reverse mutations, but now a forward mutation assay is possible. Carrie Valentine is working on a system for detection of frameshifts that seems promising. Mutations from the animal are about 1/3 of recovered spontaneous mutations fixed in vivo, with about 2/3 of mutations being ex vivo; in cell culture, the frequency of ex vivo mutations is similar to the frequency in the animals, but the spontaneous in vivo frequency is higher. After treatment, only the in vivo mutation frequency increases. He has coined the phrase "furry Ames test".
John Heddle [jheddle@yorku.ca] prompted a discussion re Big Blue,including the use of B.B. to study ex vivo transition of lesions to mutations. In cultured cells, under semi-starved conditions (John, amplify ?), no mutations are recovered. This led to a discussion on methodological issues around media composition and troubleshooting selection or color selection issues. It was agreed there are differences between lot numbers of undefined media components (e.g. yeast extract), and agar (lots and brands). John noted that Jason Bielas [jbielas@yorku.ca] has done a lot of work on these problems. Contact Jason directly by email for details. It may be useful to launch a discussion on other people's experiences and solutions. This could be done by an special edition email newsletter from TIVM, or a separate mailout. Clearly there are ongoing issues, and given the need to develop strong protocols for the international agreements on the use of transgenics, the time is now.
Jim Munroe talked about 5-meC content in transgenes, noting as have others, that events at CpG sites (of 5-meC) contribute to high background. But does 5-meC content in the transgenes correlate with mutation and tissue specificity (tissue-specific differences previously seen). He also noted the cII has a higher background relative to lacI in B.B. Is there a difference in 5-meC between tissues in B.B. mouse (ed. note) what about in cultured cells, where methylation can be modified ? Results : lacI is fully methylated in all tissues tested, and this does NOT account for the tissue-specificity of mutations. cII is partially, but not differentially methylated in different tissues, and 5-meC burden does not correlate with m.f. in lacI or cII. He noted that lacZ in mutamouse is fully methylated in all tissues examined.
The CpG site questions keep arising. The bias towards mutation at CpG sites is often reported from transgenic models. Tom Skopek talked briefly about an extension of his "Merck II" mouse work (a lacI selection construct with most of the CpGs removed), attempting a new mouse with all the lacI CpGs removed. The construct becomes highly unstable, and no stable inserts could be recovered ! (ed. note) Clearly CpG sites in the transgenics are weird and interesting, an extension of CpG weirdness in the genome. Detailed investigations might be very interesting in the trangenic systems. How about crossing Tom's CpG-null mouse with a "normal" CpG mouse?
George Douglas [george_douglas@hc-sc.gc.ca] and Veronique Thibault[veronique.thybaud5@aventis.com] talked about the OECD and IWGTP processes for transgenic animals to be accepted into the regulatory framework. Recurrent themes include 1) how do data from transgenic models provide unique perspectives or information not found with current models (e.g. NTP or Ames). 2) how are the protocols for the tests to be optimized to improve between-test consistency and international acceptability; does the existing literature support 1) and 2) ? Are there definable gaps in the data which can be resolved to improve the likelihood of acceptance ? There is a need to focus on high-priority items, such as the unique purpose of transgenic models, how many treatments (subgroups), how long is the manifestation time, how might new data modify positions ? Notably, data are lacking on negative test results (ed. note) no surprise here, negative studies hard to publish. Is there a possiblity of a summary paper for say, EMM, compiling such data from multiple labs ?RSVP to ed.
John French [french@niehs.nih.gov] is chairing a subgroup on the p53 and rasH2 models, including combined models (e.g. XPA/p53). The editor thinks he is soliciting input on these models. Correspond directly with him.
Some Useful Information :)
Proposed design for the TIVM webpages will be announced to this group before the pages go to the EMS site, and will be available on a temporary site. Watch for it. !!
All submissions / suggestions considered. email to barry.ford@drdc-rddc.gc.ca