Nature173: 686-687 (April 10, 1954)
Are Inbred Strains Suitable for Bio-assay?
Anne McLaren and Donald Michie

Grüneberg1 has directed attention to two properties of inbred strains which should counsel caution in their use for some experimental purposes

(1) They cannot be relied upon to remain constant in their heritable properties with the passage of time, and may diverge quite rapidly when split up into separate sub-strains and colonies. (2) They do not constitute phenotypically uniform material, but may, on the contrary, be strikingly more variable than the F1 hybrids between strains.

This second proposition, for which Livesay2 obtained experimental evidence in 1930, calls into question the established practice of using inbred material for the bio-assay of food factors, hormones, drugs, poisons, pathogens, etc. In such experiments precision in the estimates, and hence uniformity in the test organisms, is of the highest importance.

The characters measured in bio-assay are of the type usually termed 'physiological', whereas the examples cited by Grüneberg all relate to morphological variation. None the less the interpretation of the phenomenon advanced by Robertson and Reeves3 and by Rasmuson4 would imply that we are dealing with a general biological law which must be expected to apply no less to 'physiological' than to 'morphological' characters. "The more heterozygous individuals," write Robertson and Reeve, "will carry a greater diversity of alleles, and these are likely to endow them with a greater biochemical versatility in development. This will lead to ... a reduced sensitivity to environmental variations, since there will be more ways of overcoming the obstacles which such variations put in the way of normal development."

Exp. Stock Gm. 'Nembutal'
per cent
Mean log
response time
Antilog
(min.)
Sums of
squares
Degrees of
freedom
Estimate of
variance
I Inbred F1 hybrids "Mousery" 0.65
0.65
0.65
1.412
1.125
1.662
26
13
46
0.4896
0.1827
0.4853
13
8
13
0.0377
0.0228
0.0373
II Inbred F1 hybrids "Mousery" 0.65
0.85
0.65
1.593
2.110
2.185
39
129
153
0.8888
0.2394
0.5891
9
16
13
0.0988
0.0150
0.0453
Combined Inbred F1 hybrids "Mousery"
1.3784
0.4221
1.0744
22
24
26
0.0627
0.0176
0.0413

Two experiments on variation in a 'physiological' character have, in fact, just been published. Yoon5 has found in mice that F1 hybrids are less variable in time of vaginal opening than the parental strains. Maynard Smith and Maynard Smith6 working with Drosophila subobscura have found a decreased variability in rate of development associated with heterozygosity for a marked chromosome.

We have recently conducted experiments with mice on the variation of a character chosen to be of the type measured in bio-assay, namely, the duration of narcosis following the intraperitoneal injection of 0.10 ml. per gm, body-weight of a dilution of a proprietary preparation ('Nembutal') of pentobarbital sodium.

We compared the variability in response of samples of three stocks of mice: inbreds from the C57BL strain, F1 hybrids from C57BL female x C3H crosses, and random-bred albinos supplied by "The Mousery", Rayleigh, Essex. Unfortunately, it was not possible to include a sample of the other parental strain (C3H). The duration of narcosis was expressed as the logarithm of the number of minutes elapsing from the abolition to the reappearance of the righting reflex. The logarithmic transformation was adopted in order to eliminate dependence of the variance upon the mean, and was found to be satisfactory for this purpose.

There were indications of differences in response both between the sexes and among different litters. In order to eliminate these sources of variation, the variances were estimated from the within-sexes within-litters sums of squares.

Two similar experiments were done on two separate occasions. On each occasion the three groups of mice were tested concurrently. In the second experiment, the hybrids were given a larger dose to compensate for their greater resistance to the drug. All mice were in the age-range 4-7 weeks, and those belonging to the same group in the same experiment were always born within the same eight days. The results of the two experiments were in substantial agreement, and are set out in the accompanying table.

It will be observed that the variance of the response is about three and a half times as great in the inbreds as in the hybrids. The difference is statistically highly significant, as judged by the z-test (P<0.01). In terms of a hypothetical bioassay experiment, three and a half times as many of the inbred as of the hybrid mice would have to be used in order to obtain results of the same accuracy.

More surprisingly, although the "Mousery" mice are as expected more variable than the hybrids, they are not more variable than the inbreds. On the contrary, the inbreds are more variable by half as much again. This difference, although not formally significant, should be regarded as suggestive, since the variability of the "Mousery" mice was almost certainly over-estimated in this experiment. It was not known which of these mice were litter mates. Hence the sum of squares used to estimate their variance includes a component which was eliminated in the analysis of the other two groups, namely, that due to differences between litters.

A more clear-cut indication that genetically heterogeneous random-bred mice may sometimes be less variable than inbred material was obtained as early as 1939 by Emmens7, who compared the variability of response to oestrone of a sub-strain of the CBA inbred strain of mice with that of a random-bred mouse colony. The variance of the inbreds, derived from the slope of the probit dosage-response curve, was about two and a half times that of the random-bred mice, and the difference was statistically highly significant.

Whether random-bred material is more or less uniform in a given case than inbred will, of course, depend upon circumstances which include the particular character which is measured and the genetical composition of the stocks which are compared. As regards the latter, the issue will be largely decided by the balance between the greater genetic variability among the random-bred organisms, making for phenotypic variability, and their greater heterozygosity, making for phenotypic uniformity.

The evidence suggests that the widely adopted policy of using inbred strains for bio-assay may be a mistaken one, and that inter-strain F1 hybrids are to be preferred. Such material combines the genetic uniformity of inbred strains with the 'buffering' action against environmental variations which heterozygosity exerts.

We are in receipt of a research grant from the Agricultural Research Council.

Anne McLaren
Donald Michie

Department of Zoology,
University College,
London, W.C.I.
Feb. 11.

  1. Grüneberg. H. [see p. 674 of this issue of Nature]
  2. Livesay. B. A.. Genetics, 15,17 (1930).
  3. Robertson. Forbes W., and Reeve, E.C.R., Nature, 170, 256 (1952).
  4. Rasmuson. M., Acta Zool., 33: 277 (1952).
  5. Yoon, Chai H., Genetics, 38, 704 (1953) (Abstract).
  6. Maynard Smith, J., and Maynard Smith, S., J. Genet., 52,152 (1954).
  7. Emmens, C. W., J. Endocrinol., 1, 373 (1939).