Innovations in breeding and genotyping are helping refine and reduce the use of research animals
Transgenic models are vital to research but generating them takes work—and a considerable number of animals. Moreover, when you breed a genetically modified animals it often results in ‘surplus’ animals.
However, scientists are devising ways to both refine and reduce the use of genetically-altered animals. Sara Wells, Director of the UK’s Mary Lyon Centre, which generates mice as models of human disease and is one of the hubs for the International Mouse Phenotyping Consortium, says a number of innovations have helped reduce genotyping and breeding of animals, such as strict colony management and different breeding schemes. At the same time, gene editing techniques like CRISPR/Cas9 is, ironically, both helping reduce the number of genetically altered animals in some situations, but increasing them in others. Nonetheless she sees a lot more attention being paid by researchers to the 3Rs than 25 years ago. “I see a real difference in new researchers, for whom the 3Rs is not an additional thought but really is really central to their studies,” she says.
How are innovations in breeding and genotyping helping you meet the 3Rs objectives?
SW: There have been many technological advances in the last few years which have greatly helped us refine our breeding and genotyping. In terms of breeding, we use genetically controlled inbred lines (only bred for five generations prior to restocking to ensure that we are not susceptible to the effects of genetic drift.) We also use strict colony management regimes to reduce the number of static matings (where the male is left in so that females are kept breeding constantly) and cryopreservation to ensure that we don’t produce animals we don’t need.
On the genotyping front, the development of blastocyst and sperm genotyping has greatly reduced the number of embryo transfers we need to perform for quality assessment. At MRC Harwell we have been concentrating very hard on quality management; that is always using allelic-specific genotyping (not an assay for a generic sequence such as cre or neo).
As more research laboratories begin using CRISPPR/Cas9 to create their transgenic animals, do you think we’ll see a spike in animals being used in studies or a drop?
SW: I think in individual studies there is a great opportunity for reduction. For some alleles (such as indels and point mutations), CRISPR/Cas9 is proving much more efficient in terms of generating mice carrying these modifications. This technology will allow us to develop more refined lines with genetic alterations which are more faithfully reflecting human diseases or much more specific models for discovery research (such as new recombinase lines or cell-specific reporters). Although there is likely to be a reduction in the generation aspect of these projects, the utility of the lines will increase. However, better preclinical models or more specific genetic models are likely to be more widely utilized and laboratories and may result in a concomitant increase in animal usage.
The major development which really will have an implication and could lead to an increase in GA animals are the large human genomic projects, sequencing hundreds and thousands of human genomes around the world and the ideas developing around precision or personalized medicine. It is inevitable that this will lead to an increase in demand for genetically altered animals (of many species) in an attempt to develop experimental models for investigating the mechanisms of many monogenic and polygenic genetic diseases.
What sorts of 3R innovations do you hope to see in the next 50 years?
SW: I think there is a long way to go in the development of efficient genome editing technologies, both in terms of germ line changes and somatic changes. This has potential to impact on all 3Rs- refining existing models and inbred backgrounds, reducing the number used in generating new GA lines and better ways of making genetic changes in vitro could replace some of the in vivo work.
Along with an increase in sophistication in genome editing we also need to increase the sensitivity and reproducibility of in vivo phenotyping tests. The enormous progress in the electronics fields will allow more automated testing, in home cage environments and using telemetry; these really will increase the robustness and complexity of the data we are able to gather from GA lines.
Are the 3Rs still an important guideline for animal research today? Why?
SW: Absolutely, they are the guiding moral principles. Just because we can, does not mean we should. Everyone has a moral obligation to seek alternatives for their in vivo experiments, use the least number of animals possible (although ensuring statistical robustness) and perform their experiments by methods which cause the least suffering. It is not a right to work with animals but a responsibility we all need to take very seriously.
How do you think the 3Rs has changed animal research in the last 50 years.
SW: I can’t comment on 50 years (I am not that old!) but definitely over the last 25 years the 3Rs has become much more prominent in discussions around experimental design, scientific justifications and the development of refined methods. I still think that there is work to do in all establishments as we try to keep up to date and contribute to advances in the 3Rs.
I see a real difference in new researchers, for whom the 3Rs is not an additional thought but really is really central to their studies. This is of course aided and supported by organizations like the NC3Rs in the UK which provides training and meetings on 3Rs themes. Lastly, over the last five years a fourth R of reproducibility has been the topic of many debates and we see more efforts towards standardization, validation and robustness of testing which is very welcome.
Between 3Rs is a Q&A series created by the Charles River Laboratories’ Eureka blog and ALN Magazine to highlight the importance of the 3Rs—replacement, reduction, and refinement—as guidelines for ethical animal use in biomedical research. If you are interested in being a part of the series, contact firstname.lastname@example.org or Regina.McEnery@crl.com.