RodentMAP Screening of Three Commonly Used Mouse Strains

Comparison of Males and Females at Three Ages for C57, 129 and FVB Mouse Strains Using the Rodent MAP.

Introduction:
Many investigators that utilize mice for research and development purposes have inquired about the "normal ranges" for the analytes measured in Rules-Based Medicine's Rodent Multi-Analyte Profile (MAP). To this end, Charles River Laboratories and Rules-Based Medicine used the Rodent MAP to biochemically phenotype plasma samples from three commonly used mouse strains. Groups of 5 males and 5 females at 3-4 weeks, 6 weeks and retired breeders (7-9 months) were bled and EDTA plasma samples prepared and frozen. After shipment overnight on dry ice to Rules-Based Medicine, the samples were tested with the Rodent MAP version 1.4 antigen panel which returned 56 quantitative analyte measurements, and to the autoimmune serology panel which measured the antibody levels in each animal's plasma to 13 common autoantigens. The resulting data are included here to provide investigators with background information and to provide a general range of concentration for these analytes in mouse EDTA plasma. Charles River Laboratories and Rules-Based Medicine want to caution investigators that this information should not be used as control data for their own MAP experiments. Our experience has shown that the results for any given strain may vary given a host of variables such as environmental factors, housing conditions, stress, time of blood collection, and method of blood collection. Also please note that the concentration of these analytes may differ significantly in serum or plasma harvested using other anti-coagulants such as citrate or heparin. There is an example of these differences in the "Case Studies" section of our website entitled "Should I use Serum or Plasma for my MAP Study?".

Materials and Methods:
C57 (C57BL/6NCrl), 129 (129S2/SvPasCrl) and FVB (FVB/NCrl) mice were housed at Charles River Laboratories' facility in Wilmington, MA using standard procedures. All mice were VAF, i.e., free of known pathogens. At the assigned time points, five animals of each sex from each strain were euthanized, bled and their blood treated with sodium EDTA to a final concentration of 5 mM. Plasma was collected after centrifugation at 13,000 x g for 2 minutes and frozen at -80oC. Frozen samples were shipped by overnight courier on dry ice to Rules-Based Medicine where they were stored at -80oC in a liquid nitrogen-backed ultra cold freezer. Rodent MAP antigen and autoimmune panels were performed on the thawed samples using standard operating procedures established at Rules-Based Medicine. Results were recorded in spreadsheet format.

Results:
Results of this biochemical phenotyping can be seen in the attached spreadsheet labeled as Table 1. The Least Detectable Dose (LDD) for each analyte is listed at the top of each column. LDD is defined as the mean background value for 20 determinations plus three standard deviations. Any value below the LDD but above the Lower Assay Limit (LAL) is reported in the spreadsheet. The LAL is defined as each assay's working sensitivity in plasma as defined by the lowest concentration calibrator used for quantitation. Any value above the LDD will possess excellent precision with coefficients of variation (CV) less than 20% and more likely below 10%. Any value between the LDD and LAL is reported but is less precise than values above the LDD. Values below the LAL are reported as . For data mining, we recommend replacing the values with either a value that is 50% of the lowest value reported in that experimental set or the LAL.

For each group of five animals, the mean and standard deviation are shown. These data should only be used as a guideline of the analyte range for the 56 analytes measured in the Rodent MAP version 1.4. Clearly, there are strain, gender and age differences that become apparent when one scans these data. In addition to the STDEV, the mean values for each group including the coefficients of variation (CV) are summarized at the bottom of Table 1. As explained above, if the mean value is near or below the LDD the CV is expected to be large.

Table 2 lists the results from the autoimmune serology panel. These results are provided as an RBM ratio which is calculated as the specific signal divided by any non-specific signal generated using a set of control microspheres. There is a cutoff RBM ratio listed under each autoantigen. If the RBM ratio is above this cutoff value, then the animal is considered to be autoimmune to that particular antigen. In this experiment, none of these strains demonstrated significant autoimmunity to the 13 antigens tested.

Table 3 is a compilation of these data again listing the means without the STDEV or CV making visual comparisons more comfortable. At the bottom of Table 3 the mean with 2 standard deviations is reported for each analyte including data from all strains, genders and age groups tested. This 95% confidence interval of the data should encompass normal animals in these three strains.

Finally, we further condensed the data into Table 4 which compares just the retired breeder adult male and female animals of the three strains using the mean value for each group. First it should be noted that this testing was performed with version 1.4 of the Rodent MAP. It measured 56 antigens as compared to the 1.5 version run today which measures 59 with the addition of CD40, CD40L and MMP9.

There were a number of analytes that displayed either strain-specific or gender-specific concentrations or both. For example, the level of epidermal growth factor (EGF) was higher in the male 129 mice than the female 129s or any member of the C57 or FVB groups. If one is therefore interested in EGF, perhaps the adult male 129 is the best animal model. Male 129s also demonstrated higher levels of eotaxin, IL-6, IP-10, MCP-1, MCP-3, MDC, and VEGF than the adult 129 females. Many of these analytes were determined to be at concentrations above the LDD in the males and below the LDD in the females. This could be an important factor in choosing your experimental model.

The C57 animals were unique in their higher levels of haptoglobin for both sexes and a female-specific elevation in VCAM-1. The C57 males and females like the 129 animals had significantly higher levels of IL-1 alpha than the FVB mice. Again, this suggests that if you want to knock out or alter IL-1 alpha expression in a mouse model, you want to use a 129 or C57 rather than an FVB mouse. The C57 male mice also displayed an elevation of MCP-3 in relation to the females as did the FVB and 129 males. The FVB mice demonstrated a uniquely low level of EGF and a high level of MIP-2 for both males and females.

Conclusion:
These data were reported here as an informational tool for our current and future customers. The results are meant to be used as an example of the data produced with our Rodent MAP and as a general guide to the expected analyte concentration ranges observed in mouse plasma. It should be noted here that these are resting, normal animals that have not been challenged by disease, environment or chemical compound. In this condition, there are numerous analytes that exist below detectable levels in this normal situation. However, with perturbation, many of these analytes become highly expressed in the blood and act as powerful biomarkers to assess disease, drug efficacy, and drug toxicity.

Using only five mice per group we were able to establish the approximate ranges for these analytes. In addition, we identified several strain-specific, analyte differences that could be useful for experimental design and data interpretation. For example, if one plans to knock out the IL-1 alpha gene and look for its depletion from the plasma, the FVB mice would be a poor choice as they normally have very low levels of IL-1 alpha in the blood.

Again, Charles River Laboratories and Rules-Based Medicine want to caution investigators that this information should not be used as control data for their own MAP experiments. Our experience has shown that the results for any given strain may vary given a host of variables such as environmental factors, housing conditions, stress, time of blood collection, and method of blood collection.

If you have questions about your experimental design and how MAP testing can be optimized for your needs, feel free to contact us at 866-RBM-MAPS or by email at info@rbmmaps.com.