Could CC-Rix mice change how we think about traditional mouse models?

From the Lund Laboratory, Vaccine and Infectious Disease Division

           Scientists have relied heavily on animal models to move research forward. However, recent discoveries have caused researchers to rethink classical animal models. In the past, inbreed mouse models were the gold standard, being well characterized, yielding reproducible results and allowing for easy generation of research tools like transgenic and knock out strains. But with current advances in across many fields and lack of appropriate animal models for some diseases, dogmas are changing. Given the diversity of the human population and immune response, academics started to ask if using a clonal mouse model really was an accurate model? In recent years, the collaborative Complex-Trait Consortium decided to address this very question by creating a Collaborative Cross (CC) mouse genetic reference panel. This resource, was made to specifically model genetic diversity and interactions in a mouse model. To do this, eight founder strains of mice (five inbreed and three wild-derived stains) were chosen (see left figure). These mice cover the three major Mus musculus subspecies with 90% common genetic variation with the variation uniformly distributed across the genome. Three generations of funnel breeding to achieve genetic contributions from all eight founders was followed by at least 20 generations of inbreeding. Researchers then used the first generation (F1) progeny from crosses for experiments (designated CC-recombinant intercross or CC-RIX). These mice provide genetic diversity in a controlled manner allowing for a wide breath of phenotypic differences under genetic control.


Figure left: A representation of breeding to create the CC-RIX mice used in the paper. Briefly, 20 generations of inbreeding of the CC mice created a recombinant inbred line and crossing two of them creates a F1 CC-RIX mouse. Figure right: The variation in frequency associated with the CC-RIX (black) range compared to the B6 (red) and BALB/c (blue) baseline values. Figure provided by Dr. Graham

           In a proof of concept genetic mapping study, researchers from Fred Hutch Vaccine and Infectious Disease Division screened over 110 CC-RIXs to map the steady state immune response in these mice. This work from Dr. Jennifer Lund’s laboratory was recently published in Cell Reports. As described by Dr. Graham, the lead author of the study, “A large scale screen over the course of four years was necessary to identify the steady state immune phenotypes, which we saw contributed to a divergent response to pathogens such as WNV, SARS, flu, and Ebola. The data presented here provide a gateway for CC strain selection for future studies of cancer immunity, autoimmune conditions, and various infections because investigators can tailor selection of strains based on the frequencies of particular T cell subsets as well as their activation status, steady-state cytokine expression, and other phenotypic selection parameters.”

            In this study, spleens from three to six mice from each group were sampled for steady state T cell phenotypes using three 13-color flow cytometry panels. These results were compared to both BALB/cJ and C57BL/6J mice, which have well documented phenotypes and are commonly used for immunologic studies. Compared to the control BALB/cJ and C57BL/6J mice the CC-RIX mice sampled had a much larger range of T cell frequencies both in total and when broken down by subset (see right figure). When the researchers sampled total splenic lymphocytes, they found 16-62% to be CD3+ T cells. Comparatively, in control animals the range was 36-46%. Similar to total T cells the breakdown for CD8+ and CD4+ in the CC-RIX animals was wide with 14-65% and 27-73% of splenic T cells respectively. The BALB/cJ and C57BL/6J mice had an average around 60% for CD4+ and 11% for CD8+ T cells, lacking the diversity seen in the CC-RIX mice. A third subset of T cells, Tregs also showed the large breadth in the CC-RIX animals. The complete data set is available on ImmPort (Immport:SDY1176). T cells were further broken down into activated and antigen experience to look for genetic control of adaptive immunity. This resulted in similar results with the CC-RIX animals having a wider breadth of response.

The group also sought out a possible relationship between the number of Tregs and their suppressive effect, to test whether frequency correlates with function. These results support the hypothesis that lower numbers may be compensated by increased activation and/or suppressive capacity. Taken as a whole, the data allows for identification of quantitative trait loci (QTLs) and potential genes within the regions that could affect immune phenotype. With the uploading of all results to ImmPort the dataset can be accessed by other researchers, prompting new improved mouse models for T cell phenotypic diversity as well as genetic mapping studies. Dr. Graham said, “Our entire dataset is available on ImmPort, allowing researchers the ability to sort and select from baseline phenotypes of interest for a particular infection or disease to perform a small subset of experiments rather than a large, time- and resource-consuming phenotypic screen. We anticipate that our dataset, along with these accompanying proof-of-concept studies detailing examples of genetic mapping of immune traits of interest, will advance the use of the CC in immunology and genetic mapping studies.”

This work is supported by National Institutes of Health.

Graham JB, Swarts JL, Mooney M, Choonoo G, Jeng S, Miller DR, Ferris MT, McWeeney S, Lund JM. 2017. Extensive Homeostatic T Cell Phenotypic Variation within the Collaborative Cross. Cell Reports, 21(8), 2313-2325.