**5. Diallele crosses to genetically modified strains**

Genetically modified animals, including humanized, transgenic and knockout mouse models, have been a vital piece in uncovering genotype–phenotype associations, but they have often suffered from the same *N*-of-1 problem as above – for example, a knockout has been produced on a single genetic background, and then phenotyped. There is ample evidence that a genetic modification produced on one genetic background can have a different phenotypic effect compared to an identical modification on a different genetic background [152–165]. Expanding above this *N*-of-1 had been difficult, as each new isogenic strain had to be produced independently with a consequent near linear increase in effort. However, each of these genetically modified isogenic lines can be added into a DAX. Now, each of any of hundreds of F1 crosses is genetically defined, replicable and isogenic, but also contains one copy of the genetic modification (**Figure 1C** and **D**). Given that there are now thousands of knockout strains available (e.g. from the International Mouse Phenotyping Consortium [166, 167]), creating a DAX is a relatively cheap and quick method by which to test the effects of genetic background [158, 168–171]. By using an RI population, we can map the location of modifier loci, genes, and variants [172–174].

An excellent example of this already exists: the Alzheimer's disease BXD (AD-BXD) panel developed by Kaczorowski and colleagues [175, 176]. By crossing C57BL/6J-congenic females hemizygous for the humanized 5xFAD transgene (JAX Stock No. 008730) to males from BXD strains, they produced litters, half of which had the 5xFAD transgene (the AD-BXD), and half of which did not have the 5xFAD transgene (non-transgenic-BXD). The whole litter is genetically and environmentally identical except for the presence of the transgene, giving an immediate and directly comparable control (**Figure 1C**). By crossing the humanized 5xFAD line on a single genetic background to a diverse but defined set of BXDs, they produced a population that incorporates high levels of sequence variation mirroring that of humans. They have mapped genetic and molecular causes of cognitive loss in AD-BXD mice [154, 175–179], including a broad spectrum of cognitive loss similar to that of humans with familial and late-onset AD [177]. The human transgenes in the 5XFAD line [180] sensitizes BXD hybrids to a greater or lesser degree—some begin to lose conditioned fear memory as early as 6 months; others well after a year [175], demonstrating a gene-by-gene-by-age interaction. Variation is highly heritable and mappable and gives a powerful means by which to define genetic causality and mechanisms of memory and non-cognitive loss and resilience to loss.

Neuner et al., were also able to demonstrate 'reverse translation' from human genomic data to mouse phenotype [175]. They generated a polygenic genetic risk score using 21 human genes which increase Alzheimer's disease risk, and showed that the allele dosage was significantly associated with cognitive outcomes in the AD-BXD. This confirms firstly, that naturally occurring variation in these networks has overlapping effects in mice and humans, and secondly that gene-phenotype associations translate across species. This approach can be applied to many other phenotypes.

Given that phenotypes from genetically engineered mice on a single genetic background cannot be reliably generalized to other mouse genetic backgrounds [158], it is unsurprising that there are difficulties in generalizing to other species. By crossing genetically modified lines to RI strains to produce a DAX, we overcome this problem and allow the integration and translation of data to other populations and other species.

## **6. Integration and translation with other populations**

Compared to conventional F2s and advanced intercrosses (AIs), outcrossed heterogenous stock, or diversity outbred stock, the BXD are particularly advantageous when the heritability of a trait is moderate or low because the genetic signal can be boosted greatly by resampling isogenic members of the same line many times [38]. The drawbacks of the BXDs are lower precision, and a decreased amount of variation in the population compared to e.g. multiparent families (such as the Collaborative Cross and the Diversity Outbred), and a consequent decrease in the total phenotypic variance [181]. We consider this an acceptable drawback, as we have shown that medically relevant phenotypes have variation in the family and it is possible to achieve subcentimorgan mapping precision using only half of the full set of strains [82]. Beyond this level of precision, an efficient method to transition from QTLs to causal genes, variants, and mechanisms is to take advantage of complementary resources. These include sets of other murine mapping resources, efficient *in vitro* and *in vivo* screens [74, 132, 182], and human genome-wide association study (GWAS) data.

As a specific example of combining murine populations, Taylor's cadmium testicular toxicity mutation (BXD Phenotype 13035) that was unmappable in 1973 now maps to 3 Mb on GeneNetwork.org. When combined with SNP data for common strains, the variant can be restricted to a 400 Kb region that includes the causal *Slc39a8* gene, a heavy metal transporter expressed almost exclusively in the testes [183].

#### *Recombinant Inbred Mice as Models for Experimental Precision Medicine and Biology DOI: http://dx.doi.org/10.5772/intechopen.96173*

Mouse-to-human genetic translation has at least a 20-year history [184], but has taken off now that GWAS are routine [48, 78, 111, 112, 123, 125, 185, 186]. Human GWAS data can be used to refine QTL found in mice, e.g. taking advantage of the power to detect associations in the BXD to identify a homologous region in humans, and then using the precision of human GWAS to identify a candidate gene [185–187].

More importantly, mouse data can be used to determine the function and causal pathway for associations made in humans. Finding variant-phenotype associations for any phenotype with GWASs is now only limited by one's ability to collect phenotypes, but interpreting and determining the function of these variants is far more difficult, given the environmental and genetic variation in any human population. RI mice, such as the BXD, provide a method of 'reverse-translation', from human-to-mouse. Again, the work of Kaczorowski and colleagues above provides an excellent example [175] that can be applied to any other phenotypes.
