8. Strains of Mice and Rats

 

76% of animals used in research in the UK in 2008 were mice or rats, as sown below, and the use of mice continues to grow. But there are lots of types of these species. What are they all and what are their properties?

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strains1

The main types are

  • Inbred strains (inbred lines are called “strains”)
  • Outbred stocks (outbred lines are called “stocks”)
  • Mutants Genetically modified strains (not discussed here)


Outbred stocks

They are vigorous, cheap and prolific and are widely (and probably wrongly) used in research. They are usually maintained as large breeding colonies within which there is inter-individual genetic variation. They are maintained by random (or haphazard) mating systems. Each animal will be genetically different, but the extent of genetic variation depends on the history of the colony. Genetic bottlenecks such as when a new colony is established or the stock is hysterectomy-re-derived to eliminate disease, will tend to reduce the genetic variation, while an outcross to a different stock (sometimes by mistake) will increase it.

As research models they have some disadvantages:

  • They can change rapidly in characteristics due to selection, inbreeding and random genetic drift. The latter two can be minimised by maintaining large populations and ensuring as far as possible that each breeding male and female contributes to the next generation.
  • Outbred animals are usually much heavier than inbred ones as a result of many generations of selection for fast growth rate and large litter sizes.
  • They are “genetically undefined”. Nothing is known about the genotype of any individual in the colony unless it is specifically genotyped
  • Stock names such as “Sprague-Dawley”, “Wistar” or “Swiss” have little genetic meaning. There are no genetic markers to define them. Sprague-Dawley rats from different breeders will be genetically different. This means that historic data collected on such stocks may be unreliable.
  • There is no practical method of quality control. It is not even possible to distinguish between Wistar and Spreague-Dawley rats, the two most widely outbred rat stocks, although any stock of Wistar rats will differ from a stock of Sprague-Dawleys.

strains2The figure shows diagrammatically that each rat within an outbred stock is genetically distinct and also that two outbred stocks will  be different due to genetic sampling and selection.

 

Samples of outbred rats will be different due to sampling from a genetically heterogeneous population. The figure below shows the percent responders to a synthetic polypeptide in sequential samples of Sprague-Dawley rats from the same breeder over a period of about 18 months. Sample size was about 30 animals per group. Some of the variation (e.g. in samples 1-10) is what would be expected if response depended on a single genetic locus such as the major histocompatibility complex, where there is a high proportion of responders. However, this could not account for the low response in samples 17-20 which must have come from a different colony. An investigator would not normally be aware of such variation unless they were investigating single gene markers. Seven inbred strains were also typed and these were either 100% responders or 100% non-responders.

strains3

Data from Kunz HW, Gill TJ, III, Borland B. The genetic linkage of the immune response of poly (Glu52Lys33Tyr15) to the major histocompatibility locus in inbred rats. Journal of Immunogenetics 1974;1:277-287.

strains7Genetic heterogeneity can be seen in DNA fingerprints, as shown here, although this technique has been superseded by PCR of individual loci.

When should outbred stocks be used in research?

It is difficult to think of any controlled experiment where outbred stocks would be better than inbred strains except possibly where a particular stock happens to have some characteristic of interest not found in an inbred strain. The almost universal use of outbred stocks in toxicity testing has arisen by historical accident and has never been scientifically justified.

Geneticists use outbred stocks only when they have no alternative, or for a few genetic studies. For example:

  • An outbred stock can be used as a base population for a selective breeding experiment.
  • They are sometimes used in genetic mapping and gene association experiments where the genotypes of many individuals is recorded at many gene loci to see if  there are associations with a disease or response to an experimental treatment. But these are specialised (and expensive) studies.

For the vast majority of work the genetic background needs to be controlled by using inbred strains (or F1 hybrids) in order to minimise inter-individual variation. This was illustrated by several examples in section 6. Power and Sample size. The uniformity of the beagles compared with the random dogs meant that far fewer were needed to detect a specified signal. Far fewer inbred than outbred mice would be needed to detect differences in sleeping time under barbiturate anesthetic and the kidney weight was less variable in the F1 hybrid rats than in the outbred stocks.

As they are cheap to buy, outbred stock should be used if an experiment requires large amounts of a particular organ in order to extract a protein. They could also be used in classroom dissection.

Some scientists attempt to justify the use of outbred stocks on the grounds that “humans are outbred” which should make it easier to “extrapolate” to humans. But this is a fallacy. Humans and animals differ in many ways. We don’t insist on using animals weighing 70kg on the grounds that humans weight about that. And even if it were true that in some unspecified way it was easier to extrapolate to humans, what would be extrapolated would be a larger number of false negative results because the phenotypic variability inevitable leads to lower powered experiments.


Inbred strains

These are produced by >20 generations of brother x sister mating with all individuals tracing back to a single pair in the 20th. or subsequent generations. They are genetically stable and can not be changed by selective breeding. However sublines have arisen in most of the commonly used strains as a result of  “residual heterozygosity” (the sublines were separated before the ststrains402rain was fully inbred) and new mutations (relatively rare).

The figure illustrates the within-strain homogeneity and the between-strain differences.

There are >400 inbred strains of mice and 150 inbred strains of rats

 

Geneticists have recognised thstrains505eir value for many years:

“ In experimental medicine today....the use of in-bred genetic material...is just as necessary as the use of aseptic and anti-septic precautions in surgery"    C.C. Little 1936

“The introduction of inbred strains into biology is probably comparable in importance with that of the analytical balance into chemistry.” H. Grüneberg 1952

. "...the development of inbred strains has constituted probably the greatest advance in all cancer research." Heston (1963)

These DNA fingerprints (right) of four individuals of two rat strains show the isogenicity (each individual genetically identical) of inbred strains, but also that each strain is different

The key properties of inbred strains are:

Isogenicity All individuals within a strain are genetically identical. The same genotype can be produced repeatedly.

Homozygosity: Animals are homozygous at virtually all genetic loci. This leads to immortality of the genotype because offspring are identically, genetically identical to their parents.

Phenotypic uniformity: Genetic uniformity leads to phenotypic uniformity. This means either that fewer animals can be used or the power of experiments using inbred strains will be higher than if an outbred stock had been used.

Long-term stability: Inbred strains can not be changed by selective breeding once they have become fully inbred. New mutations will lead to gradual genetic drift so it is important for investigators to specify the sub-strains which they use.

Identifiability: Each inbred strain has a unique set of genetic markers which can be used for genetic quality control. Strains do occasionally become genetically contaminated by a non-strain mating, but this can be recognised using such markers. Investigators are advised to save some tissue or DNA from the animals they use so that if they get unexpected results they can check that the animals were what they were supposed to be.

Individuality. Each strain is unique and will be different from other strains in many ways which are likely to be of interest to research scientists. Strains differ in life-span and types of spontaneous disease, there are physiological and biochemical differences between them, and they will respond differently to drugs and chemicals.

It is a perfectly acceptable scientific strategy to work on, say, C57BL/6 strain mice, or F344 rats provided it is clearly understood that the results only apply to that strain and may not apply to other strains. In many cases it is possible to do an experiment using several strains without increasing the total number of animals by using a factorial experimental design

Background data. Many thousands of papers are published each year involving inbred mouse and rat strains. Background data on strain characteristics and mouse genetics accumulates rapidly. There are now extensive databases on mouse, and to a lesser extent rat genetics. These include:

    The mouse phenome database. This has data on a wide range of strain charactersitics, searchable by subject area (e.g. behaviour, blood, bone, development etc.), strain, intervention, study design etc.

    Mouse genome informatics. This has data on genes, phenotypes, disease models, gene expression, gene function, pathways, recombinases, strans and SNPs, tumours and orthology.

    International mouse strain resources (IMSR), a searchable database of mouse stocks and strains available world wide.

    The JAX mice database. This provides extensive information on mouse genetics, specifically relating to mice maintained by the Jackson Laboratory, Bar Harbor, Maine, USA.

    The Rat Genome Database. This provides a comprehensive database on rat genetics.

When should inbred strains be used in research?

Inbred strains should be used in all experiments using mice or rats unless the use of an outbred stock is specifically justified for a particular project..

Derived inbred strains (Only brief details are given here)

There a number of more specialised strains derived from straight inbred strains. These include:

Coisogenic strains: A pair of strains which differ at only a single genetic locus (the differential locus) as a result of a mutation. “Knockout” strains usually fall into this category. Any differences between a pair of coisogenic strains will be due to the effect of the differential gene.

Congenic strains: A pair of strains which differ at a single genetic locus plus a section of chromosome. These are produced by back crossing a mutation or polymorphism to an inbred strain. The length of associated chromosome depends on the number of back crossing generations.

Recombinant inbred (RI) strains: These are sets of inbred strains developed from an F1 cross between two standard inbred strains. They are used to determine the mode of inheritance of some measured phenotype.

Recombinant congenic (RC) strains. Like RI strains except they are produced from a back cross generation of a cross between two inbred strains.

Chromosome substitution strains. A full set of these strains would include a genetic background strain and 20 strains in which a single chromosome has been substituted from a donor strain.  They can be used to show whether there are any genes on a particular chromosome which influence a particular trait.

Twenty-four Nobel prizes since 1960 which depended on the use of inbred strains

strains8

The first inbred strain of mice was DBA, developed in about 1909 by Dr. C.C. Little, then a graduate student at Harvard University. He went on to found the Jackson Laboratory which is now the main repository in the World for genetically defined mice. These are made available to research workers throughout the World.

The properties of inbred strains which were essential in most of these studies was the genetic uniformity (isogenicity) and stability of the strains so that an identical genotype could be obtained over a long period of time, and the differences between the strains.

However, some of the Nobel prizes depended on the properties of an individual strain. BALB/c, for example, develops myelomas if injected i.p. with mineral oil. Myeloma cell lines were used in the development of monoclonal antibodies by Kohler and Millstein. Similarly, strain 129 was used in the development of embryonic stem cells by Martin Evens, and these have been central in the development of knockout strains by Smithies and Capecci. Only recently have good ES cell lines been developed from other strains.

Some mutant mice and rats
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