Laboratory rodents provide a useful model for aging processes in humans. Various genetic "types" of stock are available, including outbred stocks and inbred strains and their derivatives. Inbred strains, which can be regarded as clones of genetically identical individuals, provide a powerful research tool for studies in many disciplines, including gerontology. However, some form of genetic quality control is essential to ensure that the strains are authentic. Single gene polymorphisms, particularly those detected by electrophoresis and immunological methods, provide a powerful tool for such quality control, though these methods are expensive and require considerable expertise. Methods based on several loci studied simultaneously include skin grafting and polyvalent alloantisera. These methods are often quick and technically relatively easy, but are less flexible than the single locus methods. Methods based on DNA restriction fragment length polymorphisms (RFLPs) fall into both categories depending on whether a single locus or a multilocus probe, such as the fingerprinting probes, is used. These DNA-based methods have many advantages, and are likely to be the methods of choice in the future.

Aging↗

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Warning: the use of heterogeneous mice may seriously damage your research.

Genetically heterogeneous (GH) mice and rats continue to be widely used in research even though the case for using isogenic strains has been argued repeatedly. The paper by Miller et al. in this issue appears to be the only one in the last 22 to attempt a scientific justification for the continued use of (a limited subset of) GH stocks. However, although they are to be commended for bravery, they fail to make their case. GH stocks represent poor material for controlled studies because genetic heterogeneity normally leads to phenotypic variability and a decline in experimental sensitivity. To counter this argument, Miller et al. claim that phenotypic variability may actually be smaller in GH animals than in their isogenic parents. Were this so (e.g., all mice being short lived, small, and aggressive), it is difficult to see how the use of such a stock could increase the generality of research results based on it, as claimed by Miller et al. Isogenic strains are a vital, proven, and powerful resource for biomedical research, and should be used in preference to GH stocks by all scientists who use laboratory rodents.

Animals↗

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Experimental design and husbandry.

Rodent gerontology experiments should be carefully designed and correctly analyzed so as to provide the maximum amount of information for the minimum amount of work. There are five criteria for a "good" experimental design. These are applicable both to in vivo and in vitro experiments: (1) The experiment should be unbiased so that it is possible to make a true comparison between treatment groups in the knowledge that no one group has a more favorable "environment." (2) The experiment should have high precision so that if there is a true treatment effect there will be a good chance of detecting it. This is obtained by selecting uniform material such as isogenic strains, which are free of pathogenic microorganisms, and by using randomized block experimental designs. It can also be increased by increasing the number of observations. However, increasing the size of the experiment beyond a certain point will only marginally increase precision. (3) The experiment should have a wide range of applicability so it should be designed to explore the sensitivity of the observed experimental treatment effect to other variables such as the strain, sex, diet, husbandry, and age of the animals. With in vitro data, variables such as media composition and incubation times may also be important. The importance of such variables can often be evaluated efficiently using "factorial" experimental designs, without any substantial increase in the overall number of animals. (4) The experiment should be simple so that there is little chance of groups becoming muddled. Generally, formal experimental designs that are planned before the work starts should be used. (5) The experiment should provide the ability to calculate uncertainty. In other words, it should be capable of being statistically analyzed so that the level of confidence in the results can be quantified.

Animal Husbandry↗

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Relationships among colonies of the laboratory rat.

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Animals↗

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Vote of thanks for Dr Jon Richmond's second Annual FRAME Lecture.

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Animal Welfare↗

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Guidelines for the design and statistical analysis of experiments in papers submitted to ATLA.

In vitro experiments need to be well designed and correctly analysed if they are to achieve their full potential to replace the use of animals in research. An "experiment" is a procedure for collecting scientific data in order to answer a hypothesis, or to provide material for generating new hypotheses, and differs from a survey because the scientist has control over the treatments that can be applied. Most experiments can be classified into one of a few formal designs, the most common being completely randomised, and randomised block designs. These are quite common with in vitro experiments, which are often replicated in time. Some experiments involve a single independent (treatment) variable, while other "factorial" designs simultaneously vary two or more independent variables, such as drug treatment and cell line. Factorial designs often provide additional information at little extra cost. Experiments need to be carefully planned to avoid bias, be powerful yet simple, provide for a valid statistical analysis and, in some cases, have a wide range of applicability. Virtually all experiments need some sort of statistical analysis in order to take account of biological variation among the experimental subjects. Parametric methods using the t test or analysis of variance are usually more powerful than non-parametric methods, provided the underlying assumptions of normality of the residuals and equal variances are approximately valid. The statistical analyses of data from a completely randomised design, and from a randomised-block design are demonstrated in Appendices 1 and 2, and methods of determining sample size are discussed in Appendix 3. Appendix 4 gives a checklist for authors submitting papers to ATLA.

Animal Testing Alternatives↗

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Additional evidence that the K-ras protooncogene is a candidate for the major mouse pulmonary adenoma susceptibility (Pas-1) gene.

A locus for mouse pulmonary adenoma susceptibility, Pas-1, has been mapped on distal chromosome 6, where the K-ras gene is located. Allele-specific activation and expression of the K-ras allele from the susceptible parent has been observed in lung tumors from F1 hybrid mice. We report here genetic mapping of lung tumor susceptibility genes in urethane-treated A x B and B x A recombinant inbred (RI) mice using microsatellite markers to seek further evidence for the K-ras gene as candidate for Pas-1. The K-ras genotype differs between the A/J and C57BL/6J progenitors of the RI strains, and distal chromosome 6 contained a major lung tumor susceptibility determinant in the RI mice. Additional evidence that Pas-1 is K-ras involved linkage analysis of (A/JOLaHsd x BALB/ cOLaHsd) F2 intercross mice whose parents shared the same K-ras genotype. In contrast to the results with the A x B and B x A RI strains, no distal chromosome 6 site was significantly associated with tumor development in these F2 mice. In addition to this major locus, linkage analysis of the RI mice revealed additional quantitative trait loci for susceptibility on chromosomes 10, 17, and 19. These loci may serve as modifiers of Pas-1. The relationship between the K-ras genotype and the frequency of K-ras mutations in urethane-induced lung tumors from the RI mice was also explored. All 18 tumor DNAs from RI mice with high susceptibility contained an AT-->TA transversion at the second base of K-ras codon 61. This was also true for DNAs from 27 of 27 (100%) tumors in mice with high intermediate susceptibility. In RI strains with a low intermediate susceptibility, the DNA from 39 of 47 (83%) tumors contained an AT-->TA transversion at codon 61, and only 13 of 21 (62%) tumors had this mutation in the most resistant group. This reflects a positive correlation between the frequency of K-ras mutations in lung tumors of A x B or B x A RI strains and their susceptibility to lung carcinogenesis. Since K-ras appears to be Pas-1, these results suggest that some RI mice that have the resistant K-ras or Pas-1 allele undergo tumor development by a K-ras-independent route.

Adenoma↗

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Discriminant analysis of biochemical parameters in liver disease.

Discriminant function analysis has been used to investigate the relative value of six biochemical parameters (plasma ferritin, C-reactive-protein, bilirubin, alkaline phosphatase, glutamic oxaloacetic acid transaminase and albumin) in the diagnosis of liver disease. This was done among four groups totalling 70 subjects including healthy controls and patients with acute viral hepatitis, liver cirrhosis and primary hepatocellular carcinoma. Albumin had most value in distinguishing between groups, followed cumulatively by ferritin, alkaline phosphatase, C-reactive protein, bilirubin and glutamic oxaloacetic acid transaminase. However, if data on albumin, alkaline phosphatase, bilirubin and glutamic oxaloacetic acid transaminase had already been routinely collected, there would be no advantage in collecting data on ferritin and C-reactive protein. Any four of the six parameters would be of about equal value in distinguishing between diagnostic groups. When the data on all six biochemical parameters was combined in an optimum way, about 66% of all individuals could be correctly assigned to one of the four groups using biochemical markers alone. While the control subjects and patients with acute viral hepatitis formed a relatively well defined, tight cluster (apart from two patients with acute viral hepatitis), patients with liver cirrhosis and primary hepatocellular carcinoma were almost indistinguishable, using these biochemical parameters. If the latter two groups were pooled, then about 86% of subjects could be correctly classified.

Alkaline Phosphatase↗