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					    Linkage Mapping of
   Mandibular Length in
Recombinant Congenic Mice
                   Derek Wilson Jr.
              Fort Valley State University

           In Kyu Han, Seung Pyo Han,
     Suzanne Litscher and Robert Blank, Ph.D.
    Departments of Medicine and Endocrinology
       School of Medicine and Public Health                            Overall, it has been estimated that 50–70% of bone strength is
                                                                       determined genetically (2).
Abstract                                                               Here, we report application of the linkage mapping strategy in
                                                                       a subset of the F2 generation of a reciprocal intercross
In previous experiments we exploited the genetic structure of          between the recombinant congenic strains HcB-8 and HcB-23
recombinant congenic mouse strains by performing a                     as used in previous work by Blank lab (refer to Saless et al.
reciprocal intercross of the strains HcB-8 and HcB-23. It was
                                                                       (2009). HcB-8 and HcB-23 differ in tissue-level
found that on a short region of chromosome 4 there lies a
                                                                       biomechanical performance (2, 13) and so we therefore chose
quantitative trait locus (QTL) affecting femoral size,                 them as parents for those past experiments. In those previous
ellipticity, and strength. Ece1, encoding endothelin converting        works we found a strong quantitative trait locus (QTL)
enzyme 1, is within this QTL and is a strong candidate gene.
                                                                       affecting femoral size, ellipticity, and strength on a short
Previous work by others has also shown that endothelin
                                                                       region of chromosome 4. Ece1, encoding endothelin
signaling is necessary for normal growth of the mandible               converting enzyme 1, is within the QTL and is a strong
during embryonic development. In this experiment we used               candidate gene. Also work by Kurihara et al. (1994 and 1995)
the F2 generation of the previous experiments to explore the
                                                                       has demonstrated endothelin signaling which is shown to
correlation between the Ece1 gene and the mandibular size of
                                                                       promote bone formation. Endothelin-1 knockout mice die at
the mice. We hypothesized that since Ece1 is the gene                  birth with respiratory failure, hypoplasia of the mandible,
responsible for the femoral traits, there would therefore be           hypoplasia of other craniofacial bones, and cleft palate and
significant linkage of mandibular length to the same
                                                                       cardiac abnormalities, while heterozygous knockout mice
chromosome 4 region, and that other QTLs affecting femoral
                                                                       appear grossly normal but have elevated blood pressure
size and strength would also affect mandibular length.                 (Clines et al. (2006)). Thus we hypothesize that:
                                                                             Ece1 is the gene responsible for the femoral traits
Using QTL Cartographer, we analyzed 93 of the 603 mice in
                                                                             There will therefore be significant linkage of
the F2 generation and found QTLs on chromosomes 1, 3, and
                                                                                mandibular length to the same chromosome 4 region.
6. These coincide with QTLs affecting the femur, but we did
not find a QTL on chromosome 4, rejecting one of our                         Some QTLs affecting femoral traits will affect
hypotheses. One great limitation due to time constraints is                     mandibular length
that only about 15% of the entire cross was analyzed so the
final data may differ from what is reported here. Work to              Materials & Methods
complete analysis of mandibular length of the cross is in
progress.                                                              Mice
                                                                       The parental mice in this study were the recombinant congenic
Introduction                                                           strains HcB-8 and HcB- 23, produced by inbreeding N3
                                                                       C3H/DiSnA (C3H) x C57BL/10ScSnA (B10) mice to fixation
The Blank lab works to study the genetics of biomechanical             (4). This breeding program resulted in inbred strains harboring
performance because fractures are an important and complex             alleles of B10 origin at approximately 1/8 of the genome on a
health problem. Fracture susceptibility depends jointly on             C3H background. Therefore, only approximately ¼ of the
bone mineral content (BMC), bone mineral density (BMD),                genome segregates in an intercross of HcB-8 and HcB-23,
gross bone anatomy, and bone microarchitecture and quality.            with the remaining portions of the genome fixed for the same



                                                                  69
allele in both parental strains. We performed a reciprocal F2             Landmark Measurement
intercross and maintained the animals to an age of 17 + 1                 Using the Sigma Scan program, the distances between the
weeks, as this is the age at which mice achieve peak bone                 landmarks were measured (refer to Figure 1 and Figure 4).
mass (1). Mice were housed 2-5 mice per 500cm2 cages, with                The mental-molar distance is the separation between points 10
12h light-dark cycling, given autoclaved tap water, and fed               and 12 or 19 and 21. The mental to coracoid distance is the
laboratory rodent chow 5001 (PMI Nutrition International,                 separation between points 10 and 1 or 19 and 3. The mental to
Richmond, IN) ad lib. Animals were euthanized by CO2                      condylar distance is the separation between points 10 and 6 or
inhalation, following AVMA recommendation. Immediately                    19 and 15. The molar to coracoid distance is the separation
following sacrifice, animals were weighed and measured                    between points 12 and 1 or 21 and 3. The molar to condylar
(rostro-anal length), viscera were harvested for DNA isolation            distance is the separation between points 12 and 6 or 21 and
(Puregene), and femora and humeri were dissected free of soft             15. The coracoid to molar distance is the separation between
tissue for additional phenotyping. Bones were wrapped in                  points 1 and 6 or 3 and 15. We averaged the distances right
phosphate buffered saline-saturated gauze and stored frozen at            and left sided distances, yielding 6 distances for linkage
-70°. The University of Wisconsin and the William S.                      analysis.
Middleton Memorial Veterans’ Hospital IACUCs approved
the animal protocol (1).                                                  Linkage Analysis
                                                                          Linkage analysis was performed with QTL Cartographer (14,
Tissue Removal & Storage                                                  15) by composite interval mapping (17), using genotype data
After sacrifice, the mice skull(s) were cleaned and all soft              as previously reported in Saless et al. (2009). The mandibular
tissue was removed. On day1, the scalps were peeled back off              distances were adjusted for sex by linear regression prior to
of the muscle and removed. Any muscle tissue easily removed               linkage analysis. The significance threshold was set at an
was taken and the skulls were placed in a 1M solution of KOH              LOD score of 2.8 because this was the significance level
for 24hrs. On day 2 the skulls were rinsed and the mandibles              established in this cross for femoral traits (Saless 2009).
were disassembled. Soft tissue was then removed further. The
skulls were placed in 10ml of Tris buffer, which has a pH of 7,
for 24hrs. On day 3 the skulls were boiled to remove any last             Results
traces of soft tissue and then were stored in a solution of 1 part
glycerol and 1 part 95% ethanol.                                          The informative regions (marked in Figure 3 as red or green)
                                                                          of the HcB-8 x HcB-23 intercross span approximately 22% of
                                                                          the genome. Crossovers occurring during the generation of
Photographs                                                               these recombinant congenic strains have led to fixation of
Once the skulls were harvested and all the soft tissues were
                                                                          several small chromosome segments of C57BL/10ScSnA
removed photographs of the mandible were taken using the
                                                                          origin in either HcB-8 or HcB-23. An intercross is only
Digital BlueTM QX5TM Computer Microscope Camera. The
                                                                          informative in those chromosome regions where the parental
objective of the bone picture is to obtain a clear, focused and
                                                                          strains harbor different alleles. The known informative regions
properly aligned photograph of each bone at specific
                                                                          of our cross are limited to chromosomes 1, 2, 3, 4, 6, 9, 10, 15,
landmarks (Figure 1 and Figure 2) for further use with the
                                                                          17, and 19 (1).
Sigma Scan program.




Figure 1




                                                                          Figure 3




Figure 2

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                                                                                    Discussion

                                                                                    Due to time constraints and deadlines set on this article, data
                                                                                    from only 93 mice was analyzed and the entire data set of 603
                                                                                    mice was not gathered. Normally data would not be analyzed
                                                                                    until all the data was compiled. It should be stated that
                                                                                    because only about 15% of the data was analyzed there may
                                                                                    be differences in the complete analysis. Also, because of time
                                                                                    constraints, permutation tests to calculate the correct threshold
                                                                                    for the linkage map could not be done so the average threshold
                                                                                    for the femoral traits from similar linkage mapping done in
Figure 4. Shows a photo of a mandible after measurements with Sigma Scan            Saless et al. (2009) was used as a substitute. This is another
program have been done.
                                                                                    limitation of our experiment and it will be addressed when we
                                                                                    complete the data set.

                                                                                    In this subset of the cross, significant QTLs for mandibular
                                                                                    length are present on chromosomes 1, 3, and 6. These coincide
                                                                                    with femoral QTLs and support one part of our hypothesis.
                                                                                    There is no indication of QTLs on chromosome 4. Therefore,
                                                                                    the preliminary data supports the idea that the same QTLs
                                                                                    affect multiple skeletal sites, including both femora and
                                                                                    mandibles. However, there is no evidence that the
                                                                                    chromosome 4 QTL, which harbors Ece1, plays any role in
                                                                                    contributing to differences of mandibular length in the HcB-8
                                                                                    x HcB-23 intercross.

                                                                                    The next step in this project is to complete the mandibular
                                                                                    length data set. This work is presently ongoing in the Blank
                                                                                    laboratory.

                                                                                    Acknowledgments
Figure 5. Linkage map shows QTLs on chromosomes 1 (molar-coracoid LOD
= 4.2), 3 (mental-molar LOD = 2.9, and 6 (molar-condylar LOD = 3.3,
coracoid-condylar LOD = 3.3). No QTL is present on chromosome 4.                    I would like to thank my mentor Suzanne Litscher and my
                                                                                    principal investigator Dr. Robert Blank for all of their
              HcB-8               HcB-23            F2                              guidance and assistance throughout the summer and in the lab.
              F (N M (N F (N= M (N F (N M (N
              = 4)      = 3)      6)       = 3)     = 42)     = 51)
mental-       2.70 + 2.66 + 2.80 + 2.64 + 2.66 + 2.56 +                             Funding
molar         0.04      0.08      0.11     0.22     0.15      0.12
mental-       7.81 + 7.64 + 8.17 + 8.04 + 7.83 + 7.76 +                             IBS-SRP supported this student with funding from the:
coracoid1     0.13      0.11      0.12     0.36     0.26      0.28
mental-       11.22     11.05     11.55    11.50    11.32     11.26
                                                                                         National Science Foundation (DBI-0552806)
condyle2      + 0.17 + 0.11 + 0.17 + 0.27 + 0.23 + 0.26                                  University of Wisconsin-Madison Graduate School
molar-        5.12 + 5.01 + 5.38 + 5.42 + 5.24 + 5.25 +
coracoid3     0.15      0.16      0.11     0.15     0.18      0.19
molar-        8.53 + 8.39 + 8.76 + 8.87 + 8.67 + 8.71 +
                                                                                    The lab supported this research with funds from the:
condyle3      0.22      0.12      0.13     0.06     0.17      0.19                          Department of Veteran Affairs (Merit Award)
coracoid-     3.71 + 3.72 + 3.76 + 3.85 + 3.81 + 3.89 +                                     GRECC Service-William S. Middleton Veterans
condyle       0.16      0.22      0.11     0.06     0.22      0.13
                                                                                             Hospital
Figure 6. Projected Separations of Mandibular Landmarks
data shown in mm, mean + standard deviation. Parental Strain Differences, by
2-way ANOVA, does NOT include F2
1. Difference between HcB-8 and HcB-23 at p = 0.002, no significant
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