DATE: February 8, 2017
TIME: 1:30pm PT, 4:30pm ET

The powerful and expanding use of genetically modified mice introduces important aspects of genetics in managing these strains for breeding and experimental usage. This management takes on increasing importance within the current backdrop of focus by NIH on “rigor and reproducibility”, which means that increasingly grant and paper reviewers will expect specific data to support the stated genotype of an experimental mouse strain. We should recognize that this genotype includes both the specific genetic modification in the mice as well as the overall genetic background of these mice. The first issue is the need to genotype many of these strains for the desired genetic modification and the challenges that involves.  While genotyping of some genetically modified mouse strains is not particularly difficult, some strains can be quite challenging, and any genotyping is exacerbated when a lab is not accustomed to basic molecular biology studies.  

Specific challenges of genotyping each strain is unique to that strain but fundamentally it involves developing and utilizing a genotyping strategy that is most often dependent on polymerase chain reaction (PCR) for the assay.  Those who have worked with PCR know that each reaction has its own unique challenges of dependability that are impacted by many variables such as DNA prep, buffer, PCR machine parameters (temperature time and number of cycles), appropriate gel system to visualize the data if the assay is scored visually as opposed to a quantitative PCR.  Against the basic challenge of genotyping are unique aspects of each strain such as whether it is homozygous, hemizygous or heterozygous, and whether the modification is on a sex chromosome and whether more than one modification is involved and whether they are genetically linked in close physical proximity or unlinked on different chromosomes. A recent complexity in genotyping is the development of CRISPR knockout strains in which the mutation is different in each founder and each founder’s offspring have different challenges in genotyping.  Every strain of genetically modified mouse is unique and must be specifically genotyped appropriately every time or the genetics of the colony are disrupted and experiments fail after significant investment.  Another important issue in rigor and reproducibility is genetic background.  Genetically modified mice are made in a mixed or inbred background but for experimental reasons they are often backcrossed into other inbred backgrounds.  The genetic background has a major impact on results of many experiments.  However, genetic background is rarely directly assessed because historically it has been very difficult.  There now exists a shared resource at Dartmouth known as “DartMouse” that is under my direction, which is able to efficiently validate the genetic background of individual mice.