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Minutes, Transgenic and In Vivo Mutagenesis Interest Group, Anchorage, AK, May 2, 2002.

The interest group convened at 7:00 A.M., Thursday, May 2, 2002, in the Chart Room on the 15th floor of the Anchorage Hilton Hotel with a view of the harbor and snow-covered mountains.  Fruit, orange juice, muffins, and sweet rolls were available for breakfast. 

The chair of the TIVMIG, Carrie R. Valentine (National Center for Toxicological Research, Food and Drug Administration, USA), reported that she and the Barry Ford (TIVM secretary, Radiation Protection Bureau, Health Canada) were working on setting up a web page for the interest group.  Carrie is proposing to include short reviews of papers of interest to TIVM members that would contain a title, a one-sentence summary, and a link to a one-page review.   Any interest group member could submit a review.  The rules for submission had been sent out previously on the electronic newsletter.  There was no further discussion. 

Elections were held for the office of chair and secretary.  It was moved and seconded to retain the present slate and was approved by a unanimous vote.

Four research presentations were made, approximately 15 minutes each that concluded the meeting at 8:30 A.M.

First Speaker:  Dr. Takehiko Nohmi, Section Chief, Laboratory of Molecular Mutagenesis, National Institutes of Health Sciences Division of Genetics and Mutagenesis, Kamiyoga, Japan. 

“Mutation frequency decline in the sampling time of transgenic assays”.

Dr. Nohmi pointed out that 28 days is the current recommendation for the length of sampling time after exposure for in vivo mutagenesis mutation assays.  He presented mutational data for bone marrow from the gpt deltamouse using both 6-thioguanine selection, which identifies point mutations, and Spi- selection, which detects up to 10 kilobase (kb)-sized deletions.   Using mitomycin C (MMC) as a mutagen (1 mg/kg for 5 days, with 6,7 mice in each group), he evaluated mutants at both 1 week and 4 weeks.  The gpt mutant frequency increased 80% at 1 week, but showed no increase at 4 weeks.  Transversions were the most frequent mutation; only in the MMC-treated animals were tandem base changes found, GG>AT or TA.  With Spi- selection the highest frequency of deletions occurred at one week, and was back to background at four weeks.  Most deletions were greater than 2kb.  One week was the most appropriate time for bone marrow.  The conclusion was that for a rapidly turning over tissue such as bone marrow, the 28-day delay may be too long.

Dr. Nohmi has also produced a gpt delta rat which has several copies of the λEG10 vector integrated on chromosome 4.  The maximum mutant frequency in the liver with ENU (5 treatments of 50 mg/kg) occurred at 30 days, which is similar to that in the liver of the lacZ MutaMouse.   A:T>T:A was the most frequent mutation, which is typical for ENU.   The results suggest that the experimental conditions set for TG mice, such as sampling time, could be applicable to those for TG rats.

During discussion of this paper, it was pointed out that the standard deviation of the various time points for rat liver was so large that the greatest difference was not significant.

Another question related to whether the observed mutations could be identified as ex vivo (fixation of mutation in E. coli but not in mice).  Unlike classical lacI color selections, which allow  phages to grow in the selection plates, positive selections such as gpt, Spi-, and cII, do not allow phages or bacteria having wild-type sequences in the reporter genes to grow in the selection plates.  This substantially reduces the probability of ex vivo mutations.

Second Speaker:  Dr.HeinrichV. Malling, Senior Research Geneticist, National Institute of Environmental Health Sciences, Research Triangle Park, NC, USA. 

“Three Origins of am3 Revertants in Transgenic Cell Culture”.  

Dr.Malling presented research conducted with the reversion assay of the ΦX174 mouse (also known as the “Malling Mouse”).  He explained that although the reversion assay is not useful for mutational spectra, it is useful for kinetic studies.  Forward assay mutants always need to be sequenced, but with the reversion assay the target is always known without sequencing.   The reversion assay has been characterized by a method called “single burst analysis” in order to identify different size classes of bursts.  The reversion assay has the characteristic that one round (2 hrs at 37°) of replication is allowed in the electroporation strain of E. coli before lysis and plating on selective bacteria.  The single burst analysis is done by dividing the electroporated culture into aliquots before the 2-hour round of phage replication.  Enough aliquots are used to ensure that only 1 out of 10 aliquots contains a full-sized burst; each aliquot is plated on a different selective agar plate in order to determine the size of the burst.  Previous work had indicated that the average burst size is 185 plaque-forming units/bacterial cell. Mutations that were fixed in the animal should produce a full-sized burst, whereas those fixed in the bacterial strains would produce smaller bursts, since they were not present on both strands at the beginning of phage replication.  Previously, he had based the cut-off on the smallest burst size of a non-revertant phage, which was 30.  In this study he used mutagenic treatment to determine the smallest burst size that responded to treatment.  He did the single-burst analysis of solvent-treated and ENU-treated animals (150 mg/kg) collected 100 days after treatment in order to avoid any residual damage to DNA from the treatment.  This study indicated that the induced mutant frequency was relatively constant between bursts 15–80 plaques in size.  He identified 3 types of burst sizes that were interpreted as follows: type 1, fixed in the mouse; type 2 fixed in electroporated bacteria; type 3 fixed in the selective bacteria on the agar plates.  Only type 1 bursts (>60) responded to treatment by mutagen.  Commercial FX174 DNA has many type IIbursts. 

Dr.Malling further explained that a single dose of ENU can cause liver toxicity and result in injured cells that do not divide, but contribute recoverable transgenes.  Therefore, his most recent experiments were done in cell culture, in which the number of cell divisions post treatment was more uniform between cells.  In solvent-treated cells, only 7% of mutant plaques recovered were in type I bursts -- or, 93% of spontaneous mutant plaques were fixed in vitro.  When analyzed by mixed bursts, the mutant frequency was 0.82 x 10^-6, but when analyzed by single bursts, dropped to 0.006 x 10^-6.  The mutant frequency for the 200μg/ml-treated cells was 4.8 by mixed bursts and 4.1 by single bursts.  This resulted in a 5.9-fold increase by mixed bursts, but a 68-fold increase by single bursts.  A significant finding was that the A:T>T:A mutation, characteristic of ENU, was found only among type I revertants, i.e. the in vivo mutations.

Third Speaker: Mr. Jeffrey Wickliffe, graduate student at Texas Tech University, Lubbock, TX, USA, with Dr. Robert Baker. 

“Big Blue Mice in the Chornobyl Environment”.

Mr. Wickliffe reviewed some facts surrounding the radioactive explosion at the Chornobyl power plant in Russia (Ukraine) in 1986.  In 1992, an increased incidence of thyroid cancer was recognized followed by the observation of increased mutation rates in mtDNA and repetitive DNA elements in 1996,7.  There have also been subsequent negative studies on increased mutation rates.  He described a 90-day exposure of Big Blue mice to the Chornobyl environment in wire mesh cages in which the exposed animals received 3.3 mGy/day.  This total dose as an acute dose causes a 5-fold increase in mutant frequency.   Five animals were exposed, and 3 were placed in a control environment, unexposed to radiation.  Animals were sacrificed on the site and tissues frozen for analysis in the USA.  No significant difference in mutant frequency or mutations spectrum was observed.  These results indicate that a chronic, low dose, is less mutagenic than the same dose delivered acutely.

Fourth Speaker: Ms. Lya Hernańdez: graduate student York University, Toronto, ON, Canada, with Dr. John Heddle.

Ms. Hernańdez described difficulties that were occurring in Dr. Heddle’s laboratory with plating efficiency of plaques in the cII assay.  They had found that only one batch of peptone would support adequate plating efficiency.  During discussion, Dr. Tao Chen at NCTR, Jefferson, AR, USA, said that his cII assay was working with peptone from Sigma (St. Louis, MO, USA) and that he would assay her samples if she would send them.  Also, Dr. Heddle reported that some batches of Strategene’s packaging reaction were not effective, but had been told by Stratagene that no one else was having any problem.  He encouraged others using their products to insist on replacements if they have a similar problem.