Technical Methods

Historically, several methods of genetic quality control have ranged from the use of biochemical polymorphisms to the use of quantitative characters such as the shape of the skeleton and breeding performance.67,68 Biochemical polymorphisms are often technically difficult to determine and somewhat limited in their distribution among strains of mice and rats. Skeletal morphology and breeding performance have the disadvantage of giving a statistical result, rather than a clear-cut positive or negative answer, though breeding performance should be routinely monitored for husbandry purposes so any change should be investigated.

The development of a large number of microsatellite and other DNA-based genetic markers detected using the polymerase chain reaction (PCR) has now completely changed the situation, although changes in phenotype noticed by animal technicians or scientific users of the animals continue to be an important way in which possible genetic contamination is first identified. The great advantage of DNA-based methods is that only a small sample of tissue is needed, it can be stored indefinitely in the deep freeze, and the techniques for genotyping are essentially the same for every locus. The only differences are in the sequences of the PCR primers and possibly some minor changes in the conditions for the PCR reaction.

Microsatellites, which are the most widely used markers, are short repetitive DNA sequences with unique flanking regions. They are highly polymorphic in the number of repeats. PCR primers usually consist of about 20 base pairs of the unique flanking DNA for each microsatellite. There are many thousands of microsatellites in the mouse and rat genomes, and primers are commercially available for many of them from Research Genetics (www.resgen.com). The basic technique involves taking a sample of tissue from the animals to be tested, preparing DNA, and amplifying one or more of the microsatellites using PCR with the appropriate primers. The resulting reaction mixture is run on an agarose or poly-acrylamide gel with control samples from animals with a known genotype, where necessary. If agarose is used, alleles are usually visualized with UV light after staining with ethidium bromide, with different length alleles running different distances. If, with an inbred strain, the DNA bands are not aligned, this shows that the genotypes are not identical. Technical methods are given in many publications that use these markers, and by Litt (1991).69

There are several variants on this basic method. The system can be automated using DNA sequencing apparatus, though this is expensive and would normally only be economical if done on a large scale or if the apparatus is already available. As the bottleneck is usually running the gel, another alternative is to pool 5 or 10 samples of the PCR product in each well. Following electro-phoresis, this will result in a strong band, with satellite bands if one or more of the samples has a different genotype.

The main difficulty with genetic quality control is in deciding the number of genetic loci to use, the sample size, and sample frequency. Many microsatellite loci have more than one allele at each locus, though sometimes these can only be identified using acrylomide rather than agarose gels. Based on about 7500 comparisons, there is about a 74% chance that two unrelated inbred mouse strains will be the same at any given microsatellite locus, assuming a resolution of six or more base pairs. This means that two unknown strains should be tested at ten loci to give a 95% chance of detecting one or more differences. However, any strain or stock will normally only be at risk of becoming genetically contaminated by other strains in the same animal house, so a critical set of markers can be chosen that will detect any contamination from these strains. Usually, loci should also be chosen that are on different chromosomes to increase their statistical independence, and known alleles should differ by more than about five base pairs so that they may be easily identified.

For routine monitoring, the sample size depends primarily on the presumed extent of any genetic contamination. A high level of contamination, say above 20%, can be detected with small sample sizes, but it is virtually impossible to detect a couple of wrong matings in a colony of a thousand breeding cages. Table 9.6 shows the sample size required to detect different levels of genetic contamination at a specified level of probability. From this, it is clear that the best approach is to do everything possible to avoid contamination in the first place.

Table 9.6 Sample Size Needed to Give a 95%

Chance of Detecting Genetic Contamination for Given Levels of Contaminated Animals in the Colony

Percent Contamination Sample Size

0 0

Post a comment