GENETIC MONITORING OF LABORATORY MOUSE COLONIES IN THE MEDICAL RESEARCH COUNC(L ACCREDITATION SCHEME FOR THE SUPPLIERS OF LABORATORY ANIMALS

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1 Laboratory Animals (1974) 8, GENETIC MONITORING OF LABORATORY MOUSE COLONIES IN THE MEDICAL RESEARCH COUNC(L ACCREDITATION SCHEME FOR THE SUPPLIERS OF LABORATORY ANIMALS by MICHAEL F. FESTlNG Medical Research Council Laboratory Animals Centre, Woodmansterne Road, Carshalton, SM54EF SUMMARY A method of analysing mandible samples using 4 discriminant functions to describe mandible shape, and plotting the samples on control charts, as used in industrial quality control, is described. Repeated samples from 3 different colonies gave consistent results, with only occasional samples 'out of control'. 5 known 'illegitimate' samples were correctly rejected, and 3 out of 4 'legitimate' samples were accepted as belonging to stock LACA. In the 4th case lack of fit was attributed to subline-differentiation. This method of genetic analysis is practical for the large-scale genetic monitoring of laboratory mouse strains and stocks, and a Genetic Monitoring Scheme is being offered to commercial breeders. The Accreditation Scheme for breeders of laboratory animals was started in ] 950 with the aim of improving the quality of animals available to biomedical research workers in the United Kingdom. In 1969 a microbiological grading scheme was introduced in which each accredited colony was screened for the presence of specified pathogenic microorganisms, and graded according to the findings (Townsend 1969). The need for a method of genetic quality control has already been emphasised (Festing, ]974). The development of the 'mandible analysis' technique (Festing, 1972, 1973a, 1973b) in which strains and stocks of laboratory mice can be identified from the shape of their right mandible, has now made it possible to introduce 'genetic monitoring' of mice into the Accreditation Scheme. The genetic monitoring (GM) scheme is a voluntary one, open to all accredited breeders of mice and, in the future, other species. It makes provision for the monitoring of inbred strains, outbred stocks and F 1 hybrids. However, breeders wishing to enter the scheme must agree to abide by a number of rules in addition to those required under the microbiological grading scheme (Laboratory Animals Centre, ]974). For eample, the breeder must satisfy

2 292 M. F. FESTlNG the Centre that the correct breeding methods are being used and that adequate records are being kept. Before being eligible for the scheme the colony must consist of at least 100 breeding females. The strains and stocks must be correctly named according to internationally agreed systems of nomenclature (Staats, 1968; Festing & Staats, 1973; Festing, Kondo, Loosli, Poiley & Spiegel, 1972), and the breeder must agree to make available for publication the eact history of the colony. The breeder must also satisfy the Centre that strains or stocks of the same coat colour are maintained in such a way as to minimise the chance of an illegitimate mating. Finally, the breeder must agree to supply samples of animals for genetic analysis, as specified by the Centre. In return, the breeder will be entitled to advertise his stocks as genetically monitored under the Accreditation Scheme. MATERIALS AND METHODS Animals Samples of mice were obtained from 3 colonies known to be authentic, either because the strains were identifiable on the basis of coat colour (strain C3HjHe-mgjLac, designated C3H in this paper), or because the mice were maintained in a small closed-colony building with no other mice of the same coat colour (e.g. colony 1, Lac: LACA, designated LACA in this paper). All colonies were initially established with stock supplied by the Laboratory Table 1. Sample dates, size and means of the discriminant functions. Discriminant function sample means Colony Sample date Sample size Breeder I Feb ,06 3,30 authentic LACA Aug ,17 ],99 Oct ] ,55 ],87 Nov '50 3, ],29 Dec ],63 Jan Feb '88 3'53-2, Mar ,96 0,86 May ,75-3, Jun '06-2,41 1'27 Mar ] ],77 Mean of ] st ],61 samples Control limits upper 3,01 4,73-2, lower 0, ,30

3 GENETIC MONITORING OF MICE 293 Breeder I Aug , ,67-0'90 authentic C3H Oct ' ,28 Nov ' J'55 Dec. 72 J ,33-3,04 Jan ' , J Feb J '05-1,56 Mar , '69-3,03 May '97 J'88 -J '99-2,57 Jun ,27 3,08-1,40-2'28 Mar J,66-1'77-3,72 Mean of 10 samples -3,17 2,13-2,29-2,40 Breeder n Feb , ,99-1'00 authentic C3H Mar ,65-1,93 Apr ,04 3,14-2,05-2,03 May ,12-1,74 Jun , '66-2,19 Jul ,17 -J' Aug , '83-1'43 Sep ' ,31-0,55 Mean of 8 samples -3, '15-1,58 Overall mean for '22-2,03 C3H colonies 2 and 3 Control limits upper -2' ' lower -4, '50-3,31 Between-sampte '66 0'64 0'64 standard deviation (Festing, J974) Animals Centre. Those maintained by breeder I were established in mid and the colony maintained by breeder U in mid Samples of 9-20 male mice weighing g were ordered periodically from both breeders as shown in Table I. Average bodyweight often fell outside the ranges specified, so a correction for size was included in the statistical analysis. For comparative purposes samples of albino mice from 5 different strains or stocks obtained from various sources were included in the study to act as 'illegitimate' samples when compared with LACA, and 4 samples of what are believed to be genuine LACA mice were obtained from 4 additional breeders to act as 'legitimate' samples. Details of these additional samples are given in Table 2.

4 294 M. F. FESTING Table 2. Additional samples for comparison with LACA. Strain Sample "Mean of 4 discriminant functions size DFI DF2 DF3 DF4 Illegitimate A 4 0,38 0,36-0'59-1,55 BALB/c 9-2,08-0,30-0'64 1'65* A2G 10-1'42 0, ,60* CD-l 20 0,55* -0,70-2,51 * 0,78* CFLP 12 4,08 0,94-1, Legitimate Lac: LACA 11 2,17* 3 79* -2,37* 1-44* 3: LACA 20 1,15* 2,73* -2'50* 2,32* 4: LACA ,83* -3'85* 1,72* 5: LACA ]8 1,92* 3,63* -2'77* 1,57* These points fall within control limits for LACA given in Fig. 1. Eact details of husbandry of these colonies are not available, but in general all colonies were maintained under average to good conditions typical of the better accredited breeders. Colonies maintained by breeders I and II were rated 4-star under the Accreditation Grading Scheme, but the colonies of illegitimate and legitimate samples were maintained at levels ranging from 1- star to 4-star. There is no evidence that environment or husbandry have much influence on the characters being studied. Table 3. Coefficients for calculating* the 4 discriminant functions. Variable I Constant DFI '14 3,30-0,40-3,23 6,40-5, ,34-136,66 DF2 9,00 5, '18-1, , '73-226,83 DF3 5'04-3' '28 0,66 5'38 0'20 4'93 ] '15-216'11 DF4-0,60 4,92-6,51 0,63-0,69-8,90-2,04 ] ' ']6-0, *Where, for eample, DF, =(3-62X, +5 76X X l1 )/T where T=X, + X X ll and the Xi are the measurements as previously described (Festing, 1973a).

5 GENETIC MONITOR1NG OF MICE 295 Preparation and measurement of mandibles The mice were humanely killed and decapitated into boiling water. After about 2-3 min the flesh was sufficiently softened to allow the mandibles to be removed. These were incubated overnight in a few ml tap water with a pinch of the proteolytic enzyme papain to clean off the remaining flesh, incisors were removed, and the mandibles were dried and stored in plastic bags. A series of 11 measurements were made as previously described (Festing, 1974), and these were used to compute 4 discriminant functions (DFs) describing the shape of the mandible. The coefficients used in calculating the DFs are given in Table 3. All measurements were done personally by the author, and a programmable desk-top calculator was programmed to do all the calculations. Statistical analysis The mean and within-sample standard deviation for each of the 4 DFs was calculated for each sample. However, the standard deviation among sample means from the same colony was taken from a previous study (Festing, 1974) involving a total of 103 samples (Table 1). Sample means were used to calculate an overall mean for the colony or strain and these data were used to construct control charts as used in industrial quality control (Thirkettle, 1968). Control limits were set at 2 standard deviations each side of the overall mean. Samples were graphed in chronological order, but it should be noted that there were varying intervals between samples. Control charts are designed as a simple method of deciding whether a particular sample could have come from the same population as previous samples. If values fall within the control limits the production process (or breeding programme) may be said to be under control. However, if the values of the sample fall outside the control limits, or, if a trend is observed, or several points lie close to a control limit, then further investigation of the colony is indicated. On average, 1 point in 20 will fall outside a control limit by chance even when the colony is under control, so a single aberrant observation will not result in the colony failing its GM test, but will result in further investigation. RESULTS Means of the 4 DFs for each sample are given in Tables 1 and 2, and control charts giving plots of individual sample means for colony 1 (LACA) and both C3H colonies combined are given in Figs 1 and 2. Although there were fluctuations in the means of the samples, these were mostly well within the control limits and no consistent trends were observed. The 4th DF of the 1st sample of LACA fell outside the control limit but this was not repeated with subsequent samples. The 2nd sample of colony

6 296 M. F. FESTING 3 DF o 5 4 DF OF ~ DF o 1 I I I 10 " I Sample Number Fig. 1. Control chart for colony No.1 (LACA). Lines indicate the mean () and the control limits (X± c» for each of the 4 discriminant functions. The point falling outside control limits is circled. 2 was more seriously abnormal since 2 of the DFs fell outside the control limits. Such an abnormal sample would normally require immediate investigation. Fortunately, subsequent samples from the same colony were normal. Fluctuations between samples were not sufficiently large to obscure the differences between LAC A and C3H which were very marked. Analysis of the means of both C3H colonies showed that although most points from both colonies fell within the control limits, there were slight but significant differences

7 GENETIC MONITORING OF MICE 297 COLONY NO.2-2~ -3 D.F v )l COLONY No.3., DF o =~~ "., '". OF-3 X. y )( liz -4~ 13l o F-4 =~f, ',. ', ' ' ',, ', ' ' ~ -J,,,,,,,, I" """" Fig. 2. Sample Number Control chart for C3H from 2 differentbreeders. Points 'out of control' are circled. (at the 5 % level of probability) between them on the 1st and 2nd, but not the 3rd and 4th, DFs. Although this may have been due to environmental factors, genetic subline-differentiation between colonies separated for an estimated 6-12 generations could account for such a difference (Festing, 1973a). Comparison of Table 2 with Fig. 1 shows that none of the illegitimate samples of albino mice could have been mistaken for a sample of LACA. In the case of strains A, BALB/c and A2G, and the stock CFLP, at least 3 of the DF means fell outside the control limits for LACA given in Fig. 1. In the case of CD-l only the 2nd DF discriminated between the 2 colonies, though the degree of discrimination was substantial so that there could be little question of the sample being mistaken for a sample of LACA. In the case of the legitimate samples, 15 out of the 16 DFs fell within the control limits for LACA, and 3 out of the 4 samples would have been correctly accepted as being LACA. However, in the case of 4: LACA the 1st DF falls just outside the confidence interval. Although such a deviation could be

8 298 M. F. FESTING attributed to chance, there could also have been some genetic drift In the subline as it has been separated from colony 1 for over 4 years. DISCUSSION Any method designed for routine quality control of laboratory animals should be sufficiently simple for the bulk of the work to be done by a technician. The mandible analysis technique as originally described required the use of a computer and a good understanding of statistical methods, as well as skilled interpretation. However, the use of a desk-top programmable calculator to calculate the mean DF for a sample of mandibles, and the use of a graphical method of recording the results by use of a control chart, has considerably simplified the procedure. Preparation of the mandibles is etremely simple and requires no particular skill. Once they have been thoroughly dried they will keep indefinitely and require little storage space. Measurement is done under a binocular microscope, with the values being entered directly into the calculator, which prints out the 4 DFs automatically. These are then graphed on the control charts and, provided the points fall within the control limits (as will usually be the case), the sample is accepted. Should the sample not be acceptable because points fall outside the confidence interval, the first step would usually be to call for another sample, though other courses of action would be available. Thus, once the control charts have been devised and the calculator has been programmed, this method of genetic quality control is not unduly complicated, and could easily be handled by a technician with relatively little training. An important point that has not been considered in this paper is the size of the sample, and the frequency and method of sampling the colony. Sample sizes of 10 animals appear to give satisfactory results without undue sampling errors. Smaller samples would give poorer discrimination between strains. Larger samples would have the advantage of covering a wider percentage of the total output of a colony and reducing the confidence interval slightly, but they would take more time, and it could be argued that small samples taken frequently would be better than large but infrequent samples. Optimum sampling frequency has not been determined system mati cally, but must depend largely on the chance that an illegitimate contamination could occur. Colonies maintained in closed buildings which do not contain any other strains of the same colour, and which are maintained by approved breeding programmes, should only need to be sampled infrequently. However, if for eample 2 albino strains are maintained in the same room, the chance of an illegitimate mating could be quite high, and frequent sampling may be necessary. However, such colonies would not be acceptable under the rules of the GM Scheme. Another point that will determine the frequency of direct sampling of a colony will be the number of indirect samples obtained. Under

9 GENETIC MONITORING OF MICE 299 this scheme users might wish to submit samples of mandibles from stock that they have purchased in older to check that they have been sent animals of the correct strain or stock. It is not known whether there will be much demand for such a service, but these indirect samples could also give additional information on G M colonies. The sampling method should also be considered. Inbred strains and, in some cases, outbred stocks, may be maintained with an elite nucleus colony in which detailed records are kept and careful attention is paid to the selection of breeding stock, with one or more production colonies in which less detailed records are kept. The nucleus colony will be used both to maintain itself and to provide breeding stock for the multiplication colonies, as in the 'traffic light schemes' (Lane-Petter & Pearson, 1971). If colonies are maintained in this way samples should, if possible, be obtained direct from the nucleus colonies, since it is these colonies that have the most profound long-term influence on the colony. Unfortunately, there may be practical difficulties in obtaining such stock. Nucleus colonies are frequently small and it may be difficult to obtain a sample of 10 male mice of g. Even when stock is available it will normally be required for breeding. However, where possible in the future breeders will be urged to supply nucleus stock for the GM scheme. REFERENCES Festing, M. F. (1972). Mouse strain identification. Nature, London 238, 351. Festing, M. F. (1973a). A multivariate analysis of subline divergence in the shape of the mandible in C57BL/Gr mice. Genetical Research 21, 121. Festing, M. F. (1973b). Mouse strain idemification by mandible analysis. In The laboratory animal in drug te~ting (ed. A. Spiegel), p Stuttgart: Fisher. Festing, M. F. (1974). Genetic reliability of commercially-bred laboratory mice. Laboratory Animals 8, 265. Festing, M. F., Kondo, K., Loos]i, R., Poi]ey, S. M. & Spiegel, A. (1972). International standardized nomenclature for outbred stocks of laboratory animals. lela Bulletin 30, 4. Festing, M. F. & Staats, J. (]973). Standardized nomenclature for inbred strains of rats, 4th listing. Transph;lItation 16, 221. Laboratory Animals Centre (1974). The Accreditation and Recognition Schemes for suppliers of laboratory animals. MRC Laboratory Animals Centre Manual Series 1 (2nd ed.). Lane-Petter, W. & Pearson, A. E. G. (197]). The laboratory animal-principles and practice. London & New York: Academic Press. Staats, J. (1968). Standardized nomenclature for inbred strains of mice, 4th listing. Cancer Research 28, 391. Thirkettle, G. L. (1968). Whe!do/l's business statistics. London: McDonald & Evans. Townsend, G. H. (1969). The grading of commercially bred laboratory animals. Veterinary Record 85, 225.

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