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Genetic monitoring of laboratory strains using allozyme electrophoresis

BACKGROUND

Laboratory animals are an integral part of biomedical research, with much of this research requiring that the animals supplied are "true to label". Research projects typically cost hundreds of thousands of dollars in salary and operating budget over several years, and yet a single undetected mistake in the supply of animals may invalidate the results. Examples of the disastrous use of non-authentic animals are documented in the literature (eg. Kahan et al., 1982).

There are three possible situations where laboratory animals used by a researcher will be non-authentic. These are:-

1. The strain has become genetically contaminated by the undetected introduction into the breeding colony of one or more individuals from another strain. When this occurs in the stud line, the contamination will be perpetuated for the life of the strain. Such a contamination event may have occurred many years before an animal house acquires a strain.

2. The wrong animals are issued to the researcher due to a labelling error.

3. There are genetic differences between the sub-lines of a particular strain and the wrong sub-line is supplied for research requiring a particular sub-line to be used as an international standard.

Despite every effort to practice careful husbandry, mistakes do occur occasionally (eg escapees mating with caged animals; putting the wrong animal back into a cleaned cage etc). Moreover, sub-line differentiation is quite a common phenomenon and is itself often the result of past genetic contaminations. Clearly then there is a need to monitor the genetic authenticity of the laboratory animals supplied to researchers. This applies to any animal house where more than one strain of a species is maintained. The aim of such genetic monitoring or genetic quality control is to detect any of the above three situations. "Best practise" in laboratory animal production requires that regular genetic monitoring be part of any quality control program, and that any suspected cross-contamination is immediately investigated.

METHODS OF GENETIC MONITORING

A variety of methods are available for the genetic monitoring of laboratory animals. Traditional methods involve the use of coat colour genes, reciprocal skin-grafting, and mandible shape analysis. However, the latter two methods are difficult to apply and none of the three are applicable to all situations involving inbred and outbred strains. Moreover, many animal houses also produce a variety of F₁ crosses between different inbred strains, considerably adding to the complexity of the problem.

Since the late 1970s, the technique of allozyme electrophoresis has been the method of choice for genetic monitoring. Briefly, 20-50% of structural genes commonly exhibit genetic variation within any species. Variants at these gene loci (ie. alleles) can manifest themselves as different forms of the protein encoded by that gene. Where these different forms of the protein possess different nett electric charges they will have differing mobilities in an electric field and hence be distinguishable using the technique of electrophoresis. The proteins examined by electrophoresis are usually enzymes. Different electrophoretic forms of the same enzyme which are the product of different alleles at a single genetic locus are known as allozymes. Thus the technique of allozyme electrophoresis uses enzymes as genetic markers to obtain genetic profiles. Such genetic profiles are a fixed characteristic of an individual and can therefore be used to identify individuals, strains or species. A detailed description of the technique is given by Richardson et al. (1986) and Adams et al. (1990).

The use of allozyme electrophoresis for genetic monitoring offers three main advantages over traditional methods. These are:-

1. Greater genetic resolution Electrophoretic variation usually involves co-dominant alleles and hence it is generally possible to distinguish heterozygotes from either homozygote.

2. Convenience A large number of different gene loci are examinable in a short time and at low cost.

3. Greater power for characterizing contaminations Where a contamination event occurs, it is possible to specify the contaminating profile and hence designate the strain involved.

More recently, a large number of different DNA technologies have been able to provide additional sources of the genetic markers needed to conduct genetic monitoring for cross-contamination. Nevertheless allozyme-based genetic markers continue to be the best way of detecting cross-contamination vents between strains, for several reasons:-

1. whilst the DNA-based techniques can identify thousands of new genetic markers, the developmental work needed to produce strain profiles on the many hundreds of inbred strains and substrains in existence has not been undertaken on any marker. Genetic monitoring cannot properly proceed without these background genetic profiles.

2. many of the DNA-based techniques are more expensive than allozyme analysis.

3. some of the highly-variable DNA markers (eg microsatellites) are too variable, and are likely to show mutation-related changes in strain profile in the absence of genetic contamination.

4. the most common DNA-based technique, assessing variation in the maternally-inherited mitochondrial DNA (mtDNA), cannot detect every case of cross-contamination.

The one area where highly-variable, nuclear DNA markers offer a significant theoretical advantage over allozyme markers is for distinguishing substrains (including strains which are sublines, congenic, isogenic etc) from one another. In such cases the strains being compared are either genetically identical save for mutation or differ at only a handful of genes. However, as substrains would never be housed together in any "best practice" breeding facility, cross-contamination is generally avoided using good husbandry.

INBRED VERSUS OUTBRED STRAINS

Allozyme electrophoresis is particularly powerful for the genetic monitoring of inbred strains. Since all animals from an inbred strain are effectively homozygous for all loci and identical for all autosomal genes, then only a few individuals need be run to profile any strain. Any heterozygosity found must either be due to contamination or mutation. Heterozygosity at two or more markers indicates contamination with the "foreign" alleles indicating the contaminating source. Outbred strains present some difficulties since they will still be segregating for some loci. Nevertheless, it is still possible by screening enough individuals to determine which alleles are not present in each colony, and this information can be used as a baseline to monitor any contamination events involving that strain.

Substrains present the most difficult problem in genetic monitoring. In theory, substrains should be genetically identical to one another at all but a few loci. Thus complete genetic monitoring must of necessity involve diagnosing animals for one or more of these genes. This responsibility will usually rest with the researcher. Nevertheless, some congenic strains show allelic differences at another gene tightly linked to the key locus, thus providing a unique profile from the parent strain. The situation with transgenic and "gene knockout" strains is conceptually the same. Here routine genetic monitoring can only be used to confirm the genetic integrity of the strain background (thus ruling out cross-contamination between distinct strains as a reason for any of the experimental outcomes). The researchers using such strains should be responsible for checking the genetic authenticity of the "extra" or "missing" gene(s).

To date most allozyme monitoring has centred on the two most widely used laboratory animals, namely rats and mice. There is a large volume of published information on genetic makers in these species and detailed strain profiles are known for all major inbred strains. There is also considerable documentation available about sub-line differentiation for both species. With other species it would be first necessary to search for suitable enzyme markers before using these markers to establish strain profiles.

Genetic monitoring can use animals of any age, sex or health status. For inbred strains, it is highly desirable to save the discarded stud line and use these animals for monitoring. Where appropriate, a check on the integrity of issued animals can be achieved by randomly selecting animals from the production line(s). For outbred strains, select at random 10-20 unrelated individuals from throughout the colony. For F₁ animals, a known genetic profile for each parental strain is required.

RECOMMENDATIONS

It is recommended that suppliers of laboratory animals adhere to the following guidelines.

(1) All strains held should be screened to establish a strain profile.

REFERENCES

Adams M, van Zutphen B, den Bieman M, and Reetz IC (1990). "Biochemical techniques." in Genetic Monitoring of Inbred Strains of Rats. Hedrich HJ (ed.). Gustav Fisher Verlag, Stuttgart. (539 pp).

Kahan B, Auerbach R, Alter BJ, and Bach FH (1982). Science 217:379.

Richardson BJ, Baverstock PR, and Adams M (1986). Allozyme Electrophoresis: A Handbook for Animal Systematics and Population Studies. Academic Press, Sydney.

Details of the Evolutionary Biology Unit's Genetic Monitoring Service

The South Australian Museumâs Evolutionary Biology Unit (EBU) offers allozyme electrophoresis as a fee-for-service to animal breeding facilities and biomedical researchers requiring genetic quality control of their inbred or outbred strains. The unit has had over 20 years experience in the genetic monitoring of laboratory rats and mice throughout Australia.

The charges for electrophoretic screening cover two areas:-

The costs of allozyme monitoring are insignificant when compared to the total cost of the research programmes relying on the animals. The funds generated by this work are used to cover the costs of consumables for the project and to employ the additional staff necessary to enable us to carry out the work. The service is not intended to generate profit for the Museum.

Because many of the set-up costs for a project are largely independent of the number of animals examined (including of course writing the report), it is not possible to charge a "per animal" fee. Thus the cost per animal reduces as more animals are screened (up to a maximum of N=40).

The current charges for "on-demand" projects are:-

Sample Preparation:
$10 per tissue screened (animals sent alive*)
or $2 per tissue screened (when sent on dry ice)

Electrophoretic analyses:
Cost per enzyme marker
$ 50 (minimum cost of setting up a gel)
plus 50c for each sample run on that gel
(maximum of 40 samples per gel)

Thus a 40 sample gel will cost $70 per enzyme marker
(whereas to screen 50 samples will cost $125 per marker)

*a single $50 surcharge applies for each airport pick up

These charges are as of October, 1998 and are subject to review every two years

Discount rates apply for those prepared to ship tissue samples for screening between May 16^th - 30^th or November 16^th - 30^th of any year. These are:-

Sample Preparation: $1.50 per tissue screened
Analysis: Cost per marker = $ 40 plus 40c for each sample (maximum N=40)

Examples of charges

An animal house wants routine genetic monitoring undertaken on two ex-breeders from each of eight strains of inbred mouse (ie 16 animals for 15 genetic markers). The costs for the three types of service available are:-

(1) Discount rates - tissues taken by client and sent on dry ice via courier (can be sent at any time) for analysis during either May 16^th - 30^th or November 16^th - 30^th .

(2) On demand screen - tissues sent via courier

(3) On demand screen - animals sent alive for pickup from the airport

General Information For Clients

Routine genetic monitoring of inbred strains involves screening a standard set of 14-15 enzyme markers. Note that a separate suite of enzyme is polymorphic in rats as compared to mice, and thus each species must be run as a separate project. A minimum of two animals is required per inbred strain (preferably the ex-breeders). Additional animals (2-5) should be sent from amongst the issued animals where a genetic contamination is suspected or when a random test of the issue stock is required. Animals of any sex, age or physiological condition are acceptable. Researchers wishing to monitor outbred strains should contact EBU to discuss details of the project before proceeding with sample collection. Indeed, it is generally advantageous to discuss the aims and requirements of any screen before sending samples.

A range of tissues is usually required for genetic monitoring, since each enzyme has its own particular tissue distribution and specificity. In mice, kidney and blood are the main tissues, with liver taken routinely as a backup for special circumstances (although it is generally not used). For rats, kidney, liver, lung, and blood (separate red cells and plasma) are used, since each tissue only expresses a limited number of the polymorphic enzymes. Clients are strongly encouraged to take their own tissues to minimize costs; those wishing to do so must consult the attached protocol.

For those who still want to ship live animals, it is preferable they be delivered directly to the Museum (the company "World Couriers" provide this service). Alternatively, we can pick up animals from the airport (for which a $50 surcharge is then applied). Please address the boxes to "Mark Adams, SA Museum - Phone 82077305 Hold At Airport", and notify us 24 hours in advance of the flightâs arrival. Flights arriving after 3:00 PM are difficult to accommodate. Live animals should be shipped with enough room to move freely, and with adequate food and fluid (eg apple/carrot) to enable them to survive for at least 24 hours after we receive them.

Whether live animals or frozen tissues are involved, it is important to ship samples only after prior arrangement with the Evolutionary Biology Unit. Moreover, it is unwise to ship on a Friday; in the event of a lost consignment, it may not be possible to determine what has gone wrong until the following Monday. The most suitable days for shipping are Tuesday or Wednesday.

Allozyme analyses follow the standard principles and protocols outlined in Allozyme Electrophoresis: A Handbook for Animal Systematics and Population Studies. (BJ Richardson, PR Baverstock, and M Adams, 1986), Academic Press, Sydney. The details of the allozyme markers commonly used on rats and mice are presented in Tables 1 and 2 below. The strain profiles for most inbred strains and most allozyme markers are available in "Mouse Newsletter" and "Standardized Nomenclature for Inbred Strains of Mice", (J Staats, 1980), Cancer Research 40:2083-2138 (mice), and Genetic Monitoring of Inbred Strains of Rat. (editor H Hedrich, 1990), Gustav Fischer Verlag, Stuttgart (rats). Strain profiles not listed in the above references are available in the open scientific literature (about 50 papers) or are taken from our own survey work in this field.

For further information or advice please contact Mark Adams on 08-82077305 (email adams.mark@saugov.sa.gov.au), Malcolm Krieg on 08-82077453 (email krieg.malcolm@saugov.sa.gov.au), or Steve Donnellan on 08-82077479 (email donnellan.steve@saugov.sa.gov.au) or write/fax care of the Evolutionary Biology Unit, SA Museum, North Terrace, Adelaide, SA. 5000 (FAX 08-82077222).

Table 1. Details of allozyme markers commonly used for routine genetic monitoring in rats.

*AHD-K has not yet been formally described in the literature.
Tissue code: L = liver, K = kidney, Lu = lung, R = packed red cells, P = plasma.

Table 2. Details of allozyme markers commonly used for routine genetic monitoring in mice.

*NDPK has not yet been formally described in the literature.
Tissue code: L = liver, K = kidney, B = blood.

Protocol to provide tissues for genetic monitoring

Evolutionary Biology Unit, October 1998

Below are our requirements for tissue removal and storage for genetic monitoring. Any modifications to the type of storage container or the quantity of tissue will cause us delays in analysis; loss of tissue quality (samples arriving thawed etc) may compromise the genetic markers.

Mice: (3 tubes)

• kidney - 1 whole kidney, trimmed of all fat and cut into 2 portions

• whole blood - 0.1ml to 0.3 ml, taken using anti-coagulant

• liver - one thumbnail-sized piece (maximum of 0.3g), cut into two portions

Rats: (5 tubes)

• kidney - ~¹ of 1 kidney (maximum of 0.3g), trimmed of all fat and cut into 2 portions

• liver - one thumbnail-sized piece (maximum of 0.3g), cut into two portions

• lung - one fingernail-sized piece (maximum of 0.2g), cut into two portions

• red cells - the centrifuged pellet from 0.2-0.4ml of whole blood (with anti-coagulant)

• plasma - removed from the whole blood after centrifugation (~3,000g for 5 mins)

After their removal from ethically-euthanased animals, tissues should immediately be frozen (-20^oC or lower), and not allowed to thaw prior to their arrival at the Evolutionary Biology Unit. Frozen tissues should be sent on dry ice inside a "6-Pack" esky (fill to the top with dry ice) which is then enclosed within a cardboard box, and addressed to:-
Mark Adams,
Evolutionary Biology Unit,
South Australian Museum,
North Terrace
Adelaide SA 5000.

Companies such as Ansett Air Freight, Kwik-as-Air, Comet, World Couriers etc will pick up and deliver frozen material door-to-door (the "Overnight Economy" rate generally offers the cheapest suitable option). Details of the arrival day, carrier, and consignment number (in case a shipment goes astray) should be FAXed or phoned through to us (as packages are delivered to a communal delivery area, we need to know when to go looking).

For further information or advice please contact Mark Adams on 08-82077305 (email adams.mark@saugov.sa.gov.au), Malcolm Krieg on 08-82077453 (email krieg.malcolm@ saugov.sa.gov.au), or Steve Donnellan on 08-82077479 (email donnellan.steve@saugov.sa.gov.au) or write/fax care of the Evolutionary Biology Unit, SA Museum, North Terrace, Adelaide, SA. 5000 (FAX 08-82077222).