My first foray into research was as an undergraduate tech studying how environmental pollutants like dust, ozone, and smoke affected lung function and development (see for example, Kott et al, 2002, J Appl Physiol). My project was to measure changes in airway structure from stained tissue sections. The majority of those minimum wage hours (starting well after tissue collection, tissue processing, and paraffin embedding) went a little like this:
Never mind that my data did not make it into the previously mentioned paper. Despite my efforts, there was no difference in smooth muscle volume between the two groups I was testing.
Occasionally when a researcher contacts me in JAX Technical Information Services (TIS) for breeding advice, I recall those days on the bench where I worked my tail off trying to find a difference that didn’t exist; I had nothing to show for all my hard work except a sore wrist and a very sore attitude. In TIS, we answer questions from mouse researchers like “should I backcross my cre and lox mouse strains to a pure genetic background first and then make the knockout? Or should I create the knockout on the mixed genetic background first, gather some preliminary data, and then backcross?” I usually argue for the latter: do they want to spend years backcrossing before finding out that their hypothesis is wrong or their mutation is lethal?
Now imagine if a knockout mouse model for your favorite gene just magically landed in your lab. Imagine that it’s already on a pure, commonly used genetic background (making control selection a breeze). Imagine knowing ahead of time whether it is embryonic lethal, infertile, or has defects in major organ systems. Think of all the time and resources you would save in this scenario!
As it happens, an international consortium of geneticists is making this dream a reality. Members of the International Knockout Mouse Consortium (IKMC) and the International Mouse Phenotyping Consortium (IMPC) have embarked on a massive endeavor to systematically knock out every gene in the mouse genome, to characterize the mutants, and to make all of the materials and data publicly available to researchers worldwide. A collection of their initial findings looking at embryonic lethal genes from the first ~1800 genes completed thus far was published in the September 22, 2016 issue of Nature.
A reverse genetics approach of this scale could generate upwards of tens of thousands of new hypotheses for researchers worldwide. Given the 20,000+ genes in the mouse genome, this task would be impossible to achieve without collaboration between nearly 20 institutes around the world, including The Jackson Laboratory, and implementation of precise, high-throughput technologies, such as high-resolution 3D imaging of embryos and CRISPR/Cas9 mutagenesis to generate new knockout models. Considering the amount of time it would take to analyze a handful of histology sections or to generate knockout mice using conventional methods, the implementation of newer, high-throughput technologies will certainly expedite data collection and improve data accuracy.
Phenotypic characterization of the mice produced by this project started just a few years ago, with many more live, mutant mice and data coming through the pipeline. The wealth of information that has already been generated has the potential to impact your research directly and immediately. Below are some ways you can use this resource now, even if mice that carry mutations in your favorite gene are not yet available, or are only partially characterized.
Examples of targeting vectors generated by IKMC. When a mouse mutant for your favorite gene (tm1a) is bred to a Flp mouse strain (green), the FRT sites recombine to generate a conditional allele (tm1c). Alternatively when tm1a is bred to a Cre strain (red), the loxP sites recombine to generate a combination knockout/lacZ reporter allele (tm1b).
The phenotyping pipeline shows the data you may expect for each time point, and can serve as a resource if you are looking for a protocol to characterize your own mutants.
Access data, such as 3-D images of embryonic lethal mutants in the IMPC database. This particular online tool allows you to simultaneously dive through WT (left) and homozygous mutant (right) embryos in 3 anatomic orientations. Exporting data from several embryos into other digital analysis programs can allow you to accurately measure morphometric differences across multiple organs in an entire embryo. Carpal tunnel, be gone!
Thank you to our JAX researchers, particularly Steve Murray, Ph.D., Jim Denegre, Ph.D. and Candice Baker, Ph.D., for their contributions to the IKMC, IMPC, and to this blog article.
Dickinson ME, et. al. High-throughput discovery of novel developmental phenotypes.
Nature. 2016 Sep 14;537(7621):508-514.
IMPC website to access the standardized data: