Experiment
The wild-derived mouse strain MSM/Ms (MSM) exhibits higher total activity in the home cage than does C57BL/6JJcl (B6), a commonly used laboratory strain. Previously (J:165970) consomic strains established from crosses between B6 host and MSM donor strains were used to identify QTL influencing the differences in home cage activity between the two strains. Five QTL were identified linked to higher total cage activity than B6 in 5 consomic strains for Chr 2 (Hcga1), Chr 3 (Hcga2), Chr 4 (Hcga3), Chr 13 (Hcga8) and Chr 14 (Hgca9). In contrast, comsomic strains of Chr 6C (Hcga4), Chr 7 (Hcga5), Chr 9 (Hcga6), Chr 11 (Hcga7) and Chr 15 (Hgca10) were all less active than B6.
In the present study QTL mapping analysis was conducted using F2 mice derived from a cross between C57BL/6JJcl and consomic C57BL/6J-Chr 6.1MSM/Ms mice. [C57BL/6J-Chr 6.1MSM/Ms mice were referred to in text as B6-Chr6C, a consomic strain carrying the proximal side of Chromosome 6, a 59.6 cM chromosomal segment from the centromere to D6Mit12 derived from MSM (Fig 1a)].
Home cage tests were performed on 174 male F2 progeny at 8-16 weeks of age. Tests consisted of recording individual home cage spontaneous activity from 600 hours on the day after transfer for 3 days using an infrared sensor located above the lid of each cage. Genomic DNA was isolated from tails and the genotype of each mouse at each microsatellite marker was determined.
To analyze the locations of QTL linked with total activity, composite interval mapping based on a multiple QTL model was conducted. For composite interval mapping, model 6 was used as a standard and a forward regression method with 5 control markers and 0.5 cM increments with a window size of 10 cM. LOD scores were calculated as 4.605 likelihood ratio = 1 logarithm of odds. To determine the 5% level of significance, 3000 replications of a permutation test were carried out.
In the QTL analysis of the F2 intercross between C57BL/6JJcl and C57BL/6J-Chr 6.1MSM/Ms mice composite interval mapping identified a single QTL linked with total activity that mapped between D6Mit132 and D6Mit55 (17.6 Mbp) with a LOD score of 5.0. The mapping confirmed the previously identified QTL Hcga4 (home cage activity 4) mapped (J:165970) on Chromosome 6 using the same consomic mapping population.
To show the effect of the QTL on the total activity directly, a subconsomic strain B6.MSM-(D6Nig92.7-D6Mit374)Ms with a chromosome fragment that spanned the region (41.3 Mbp) including D6Mit132 and D5Mit55 derived from MSM was created. The subconsomic exhibited significantly lower total activity than B6 (p=0.0104) suggesting the existence of a gene for the reduced total activity in the region between D6Mit132 and D6Mit55.
Next a series if congenics were created from B6.MSM-(D6Nig92.7-D6Mit374)Ms to more precisely map the QTL. The results showed that four strains, B6.MSM-(D6Nig92.7-D6Mit256)/Ms (C7-4), B6.MSM-(D6Nig92.7-D6Mit55)/Ms (C7-15), B6.MSM-(D6Nig92.7-D6Nig108.6)/Ms (C7-24) and B6.MSM-(D6Nig100.2-D6Nig108.6)/Ms (C7-31), as well as parental strain C57BL/6J-Chr 6.1MSM/Ms and subcongenic B6.MSM-(D6Nig92.7-D6Mit374)Ms exhibited significantly lower activity than B6 (p<0.05).
In contrast, one strain, B6.MSM-(D6Nig108.6-D6Mit104)/Ms (C7-1), showed significantly higher activity than B6, but another fragment that covered the entire region of C7-1 did not. Given that the relationship of total activity and the regions specifically associated with each of the congenic strains varied among strains, it was difficult to map the QTL precisely.
Next, to identify chromosomal regions that influence the total activity more precisely, a regression model in which the decreasing and increasing effects in the chromosome regions partitioned into different strains are summed over the entire strain was used. From the results QTL regions related to total activity were mapped implying that there were four chromosomal regions where the p-values were significant (p<0.05).
The regression coefficients in those regions suggest that strain C57BL/6J-Chr 6.1MSM/Ms contains loci that decrease the total activity in the following chromosomal regions:
QTL Hcga4a (locus 1) mapped adjacent to the marker D6Nig100.2M and/or between D6Nig100.2M and D6Nig102.1M (locus 1), p=0.00312;
QTL Hcga4b (locus 2) mapped between D6Mit36 and D6Mit105, p=0.0370;
and QTL Hcga4c (locus 3) mapped between D6Mit104 and D6Mit23, p=0.0378.
In addition, it is likely to be that there is a QTL, Hcga4d, that increases the total activity in the region adjacent to the marker D6Nig108.6M (locus 4), p=0.00123. The levels of influence of these chromosomal regions on total activity can be estimated from the values of the regression coefficients.
To confirm the positive effect of locus 4, further congenic strains were established and their total activity analyses. Two strains, B6.MSM-(D6Nig30-D6Nig28)/Ms (C7-1-163) and B6.MSM-(D6Nig29-D6Mit104)/Ms (C7-1-298), were established from C7-1, and a third strain, B6.MSM-(D6Mit105-D6Nig28)/Ms (C7-5-325), was established from C7-5. One-way analysis of variance showed the significant main effect of strain on the total home-cage activity (F(4,109)=7.67, P<0.0001).
C7-1-163 showed higher total activity than B6, at a level similar to that of C7-1, but C7-1-298 showed no significant increase, which indicated that the region between D6Nig32 and D6Nig29 has an effect of increasing total activity. However, when the region extends towards the proximal side up to 4.1 Mb in C7-5-325, the congenic strain does not exhibit increased total activity.
These results suggest that the region between D6Mit36 and D6Nig30 has the effect of suppressing the increasing effect for total activity caused by a factor located in the region between D6Nig32 and D6Nig239. These results are consistent with the results of the regression analysis in that Hcgba4d, which increases the total activity, is
mapped around D6Nig108.6, and Hcga4b, which decreases it, is mapped in the region between D6Mit36 and D6Mit105.
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