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- Materials and methods
Five strains of mice commonly used in transgenic and knockout production were compared with regard to genetic background and behavior. These strains were: C57BL/6J, C57BL/6NTac, 129P3/J (formerly 129/J), 129S6/SvEvTac (formerly 129/SvEvTac) and FVB/NTac. Genotypes for 342 microsatellite markers and performance in three behavioral tests (rotorod, open field activity and habituation, and contextual and cued fear conditioning) were determined. C57BL/6J and C57BL/6NTac were found to be true substrains; there were only 12 microsatellite differences between them. Given the data on the genetic background, one might predict that the two C57BL/6 substrains should be very similar behaviorally. Indeed, there were no significant behavioral differences between C57BL/6J and C57BL/6NTac. Contrary to literature reports on other 129 strains, 129S6/SvEvTac often performed similarly to C57BL/6 strains, except that it was less active. FVB/NTac showed impaired rotorod learning and cued fear conditioning. Therefore, both 129S6/SvEvTac and C57BL/6 are recommended as background strains for targeted mutations when researchers want to evaluate their mice in any of these three behavior tests. However, any transgene on the FVB/NTac background should be transferred to B6. Habituation to the open field was analyzed using the parameters: total distance, center distance, velocity and vertical activity. Contrary to earlier studies, we found that all strains habituated to the open field in at least two of these parameters (center distance and velocity).
During the past decade, knockout and transgenic mice have offered great insight into basic biological mechanisms. More and more of these strains are continually being distributed from centralized repositories (Mutant Mouse Regional Resource Centers [MMRRC, http://www.mmrrc.org], European Mouse Mutant Archive [EMMA, http://www.emma.rm.cnr.it], Jackson Laboratories [http://www.jax.org] and Taconic Farms [http://www.taconic.com]). When describing the phenotype of these knockout and transgenic mice, researchers frequently include behavioral analyses. However, behavior is strongly influenced by genetic background, which makes it difficult to interpret the results of these behavioral findings. In gene targeting experiments, 129 strains are almost exclusively used, whereas C57BL/6 is often used for backcrossing at a later stage of the experiment. For pronuclear microinjection, FVB and C57BL/6 are frequently used. Transgenic mice made in FVB are often backcrossed to C57BL/6. Thus, behavioral characterization of the 129, C57BL/6 and FVB strains is vital for interpretation of the behavior of knockout and transgenic mice. Furthermore, as there are a number of different suppliers of these strains, it is imperative to determine, via genetic microsatellite analysis, their relatedness. Data on genetic polymorphisms among substrains are scarce for the 129 strain, and do not exist for the different C57BL/6 substrains. Thus, evidence of genetic divergence among substrains is important for interpretation of behavioral analysis of transgenic and knockout mice.
We therefore set out to characterize the genetic background and behavior of five inbred strains of mice (129S6/SvEvTac (formerly 129/SvEvTac, Festing et al. 1999), 129P3/J (formerly 129/J), C57BL/6NTac, C57BL/6J and FVB/NTac). A set of microsatellite markers was analyzed for size differences. For the behavioral characterization, we chose three tests that can be run at relatively high throughput, are at least partly automated and cover a wide variety of behavior parameters. We used the rotorod for testing motor performance and motor learning (Crawley 2000; Dunham & Miya 1957), an open field activity monitor for measuring exploratory behavior (Bolivar et al. 2000) and an automated monitor to measure contextual and cued fear conditioning behavior (Bolivar et al. 2001).
In this study, genetic and behavioral data were directly compared for the first time, the use of multiple parameters of open field behavior was introduced into inbred strain comparisons, and it was shown (contrary to earlier literature reports) that all mouse strains tested habituate to an open field, given enough exposures. We also reported on the performance of C57BL/6NTac and FVB/NTac, which had not been tested previously. Together our genetic and behavioral investigations provide a unique opportunity to compare the relationship between genes and behavior in the inbred mouse strains most frequently used in knockout and transgenic research.
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- Materials and methods
In this paper, we examined the genetic and behavioral diversity of five inbred mouse strains commonly used in transgenic and knockout production. Based on our data, the five strains: 129P3/J, 129S6/SvEvTac, C57BL/6J, C57BL/6NTac and FVB/NTac, can be grouped into three genetic groups: 129, B6 and FVB. The microsatellite data (see http://www.taconic.com for entire dataset) obtained in this study will be useful for accelerated backcrossing experiments and for the quality control of inbred lines. Whereas the genetic divergence of the 129 strains (129P3/J, 129S6/SvEvTac) found in our study supports previously reported data (Simpson et al. 1997), nothing has been reported on the relatedness of substrains of C57BL/6. Our data demonstrate that C57BL/6J and C57BL/6NTac are true substrains, and that neither has been accidentally crossed to other inbred mouse lines. Even after separate breeding for 148 generations, only 12 microsatellite polymorphisms were detected. The two strains differ by only two or four base pairs at these loci, which is consistent with single mutation events during which the DNA polymerase ‘slipped’ by one or two repeat units during replication. Assuming that 25% of all spontaneous mutations are fixed in a brother/sister mating scheme, we calculated a microsatellite mutation rate of 4.6×10−4 per generation, which is within the range reported in the literature (Brinkmann et al. 1998; Ellegren 2000; Yue et al. 2002). Given the microsatellite data, we asked whether there are similar differences in behavior, with the two B6 strains being more similar than other strains.
In the rotorod test, we distinguished between initial performance and motor learning. We found no significant strain differences in initial performance. However, female mice performed better than males overall, which is consistent with previous studies (McFadyen et al. 2003; Tarantino et al. 2000). In contrast to previous studies (Brown et al. 2002; Cook et al. 2002; McFadyen et al. 2003), we found that weight had only a marginal effect on rotorod performances. These apparent inconsistencies may be the result of protocol and apparatus differences across laboratories, as parameters such as the diameter and material covering the rod, speed and rate of acceleration of the rod and the number of trials can all have large effects on rotororod performance (Rustay et al. 2003a; Rustay et al. 2003b). In agreement with previous findings (Cook et al. 2002; McFadyen et al. 2003; Tarantino et al. 2000), all of the inbred strains learned the rotorod task, i.e. they significantly increased latency to fall over trials. However, there were strain differences, with FVB mice displaying less motor learning than other strains. There were no significant differences in motor learning between the two B6 strains.
Activity levels in the open field found in this study are generally compatible with published data (Bolivar et al. 2000; Paulus et al. 1999): FVB/NTac mice were highly active and 129S6/SvEvTac were less active than B6 mice. However, FVB/NTac mice were more active than previously reported for another substrain, FVB/NJ (Bolivar et al. 2000), even though the two substrains have been separate for only about 55 generations (Taconic Farms 2003). Further study is needed to investigate whether there is a significant difference between the two substrains, or whether this is due to different experimental conditions. Female 129P3/J mice were also more active than reported previously (Cook et al. 2002), and female C57BL/6J were more active than males. It is interesting to note that those two strains were bred at The Jackson Laboratory and went through six weeks of quarantine before being tested, which may have influenced results. There is always the possibility that environmental differences prior to testing could be at least partially responsible for behavioral differences seen in this study. Although Crabbe et al. (1999) reported that different ‘shipping histories’ of mice produced ‘almost no effects’, the potential for effects due to extended quarantine need further study.
When habituation to the open field during the four test sessions was analyzed, we found that habituation of total distance, percentage of distance traveled in the center and velocity did not covary. Therefore, one might speculate that habituation to a new environment is a complex process. Parallel to a decrease in true exploration, a habitual activity pattern is established, which includes constant or even increased activity on the margins of the open field, while the center is visited less, and the speed of movement during excursions increases as well as the time the animal spends resting. In a natural habitat, the animal would use this habitual activity to check periodically whether new food sources or mates appeared in the territory. The activity within a small area such as the one used in this experiment would constitute only a very small part of the mouse's daily routine. Open spaces would be avoided for fear of predators. This hypothesis would lead to a number of predictions, e.g. that activity should reach a plateau after a number of days, that center activity and its decrease over time should be correlated to measures of anxiety, that path stereotypy should increase over time, etc. Further studies varying these parameters might provide interesting information.
In both contextual and cued fear conditioning, even though all five inbred mouse strains displayed activity suppression, there were inbred strain differences. The strain differences of contextual and cued fear covaried, FVB/NTac reacted least and 129S6/SvEvTac reacted most strongly in both tests. This general pattern has been reported in other studies (Bolivar et al. 2001; Cook et al. 2002). However, there are important differences in the response to tone and context. Mice in this study responded less strongly to both context and cue than in a previous study using the same apparatus (Cook et al. 2002). As we ran the test at a very low light level (less than 0.1 lux) this may have reduced the response, but may also have made differences in the reaction to context more prominent. Thus, we once again find that small protocol differences can have significant effects on interlaboratory reliability, even when the same apparatus and basic protocol are used.
Generally, there were significant differences in performance in all three behavioral tests. FVB/NTac mice learned poorly on the rotorod and in the contextual and cued fear-conditioning paradigm. They habituated to the open field by increasing margin activity and decreasing center activity. 129S6/SvEvTac mice learned well on the rotorod and in the fear paradigm while having low open field activity. C57BL/6J and C57BL/6NTac mice showed good rotorod learning as well, reacted less in the contextual and cued fear test and had medium to high open field activity. Thus, performance in the three tests was not very tightly correlated, but high activity seemed to reduce the context and cued fear responses. This may be due to motivational or general activity level differences.
129S6/SvEvTac and both B6 strains appear to be equally well suited for the behavior screens described in this paper. 129S6/SvEvTac is also a relatively good breeder (Taconic Farms 2003). When a knockout mouse is developed on this 129 strain, it appears advisable to keep a population of knockout mice with pure 129S6/SvEvTac background and to start phenotype testing with this population. However, it would be prudent to start backcrossing to other strains in parallel to these experiments, because some experiments may require a different genetic background. Also, data from two different genetic backgrounds would allow for more general conclusions about the actual effects of the target gene, as well as provide information about any modifier genes.