Genetic Technologies for Building a Better Mouse

Downloaded from genesdev.cshlp.org on September 29, 2021 - Published by Cold Spring Harbor Laboratory Press REVIEW A most formidable arsenal: genetic technologies for building a better mouse James F. Clark,1 Colin J. Dinsmore,1 and Philippe Soriano Department of Cell, Developmental, and Regenerative Biology, Icahn School of Medicine at Mt. Sinai, New York, New York 10029, USA The mouse is one of the most widely used model organ- scribing Mendelian inheritance for mouse coat color char- isms for genetic study. The tools available to alter the acteristics (Cuénot 1902). Mendel himself had started mouse genome have developed over the preceding decades down this same road fifty years earlier, only for his bishop from forward screens to gene targeting in stem cells to the to snuff out the nascent breeding program because a monk recent influx of CRISPR approaches. In this review, we had no business, frankly, breeding (Paigen 2003). first consider the history of mice in genetic study, the Following Cuénot’s papers (Cuénot 1902), genetic re- development of classic approaches to genome modifica- search using the mouse began in earnest, often through tion, and how such approaches have been used and im- the study of spontaneous mouse mutants. One line of re- proved in recent years. We then turn to the recent surge search further explored the complicated genetics of pig- of nuclease-mediated techniques and how they are chang- mentation and the sometimes-surprising coincident ing the field of mouse genetics. Finally, we survey com- phenotypes. The Dominant white spotting allele W (an al- mon classes of alleles used in mice and discuss how lele of the receptor tyrosine kinase Kit) was found to affect they might be engineered using different methods. not only pigmentation but also hematopoiesis and fertili- ty, for instance. Another line focused on the genetic basis of other variable traits such as histocompatibility, sex de- termination and X-inactivation, and classic studies of tail length and the T locus (Silver 1995). Perhaps the most con- The origins of mice as a genetic system sequential endeavor, however, was the effort to understand the relationship between genetics and cancer, as The house mouse, Mus musculus, has been a tool of genet- this led to the establishment of many of today’s inbred ic inquiry for well over a century. A case can be made that mouse strains as well as the founding of mouse genetic re- the tradition dates back even further to 18th century Japan search centers such as the Jackson Laboratory in 1929, and China where local mouse and rat fanciers wrote guide- Oak Ridge in 1943, MRC Harwell in 1947, and the Nation- books on how to raise these animals, including detailed al Institute of Genetics in Japan in 1949 (Silver 1995). breeding strategies to obtain particular coat patterns Alongside research using laboratory inbred strains, study (Tokuda 1935; Kuramoto 2011). The hobby of mouse fan- of wild mouse populations led to important insights in im- cying was exported to England and from there to the Unit- munity, cancer, and adaptation (Phifer-Rixey and Nach- ed States, where fancier stocks formed the basis of many man 2015). Modern genomic techniques now couple modern inbred strains (Morse 1978). Nevertheless, the wild populations’ variations and selective pressures with first genetic experiment using mice may have been per- molecular precision (Harr et al. 2016; Barrett et al. 2019). formed in 1887 by August Weismann, who surgically re- A full accounting of the history of mouse genetic research moved the tails of mice, bred them, and found offspring is beyond the scope of this article, but has been the subject with tails of normal length (Weismann 1889). This result of several excellent books and reviews (Russell 1985; Sil- challenged Lamarckian inheritance and led Weismann to ver 1995; Paigen 2003; García-García 2020). propose the germ theory of inheritance, which stated that Even with these impressive genetic bona fides, the true hereditary information was transmitted only through potential of mouse genetics lies in the ability of the re- germ cells: the sperm and egg. The origin of mice as a mod- searcher to modify the mouse genome. This ability, along el system for genetic analysis, with intrinsic variable and with the fact that mice are mammals with substantial ge- heritable traits worthy of study, dates to 1902 when netic similarity to humans, has made mice a favorite Lucien Cuénot published the first in a series of papers de- © 2020 Clark et al. This article is distributed exclusively by Cold Spring [Keywords: gene targeting; CRISPR; mice; genetics; transgenesis] Harbor Laboratory Press for the first six months after the full-issue publi- 1These authors contributed equally to this work. cation date (see http://genesdev.cshlp.org/site/misc/terms.xhtml). After Corresponding author: philippe.soriano@mssm.edu six months, it is available under a Creative Commons License (Attribu- Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.342089. tion-NonCommercial 4.0 International), as described at http://creative- 120. commons.org/licenses/by-nc/4.0/. 1256 GENES & DEVELOPMENT 34:1256–1286 Published by Cold Spring Harbor Laboratory Press; ISSN 0890-9369/20; www.genesdev.org Downloaded from genesdev.cshlp.org on September 29, 2021 - Published by Cold Spring Harbor Laboratory Press Building a better mouse system for understanding the genetic underpinnings of been used to better understand topics ranging from mouse mammalian development and human disease (Rosenthal development (Kasarskis et al. 1998; Nolan et al. 2000), to and Brown 2007). Indeed, as of July 28, 2020, there were circadian rhythms (Vitaterna et al. 1994), congenital heart 64,204 mutant mouse alleles listed in the Mouse Genome disease (Li et al. 2015), and the genetic basis of sleep Informatics (MGI) database. The tools available to gener- (Miyoshi et al. 2019), among many others. ate these alleles have been steadily expanded from forward The precursor to mouse genetic screens arose from an genetic screens, to viral and gene trap-mediated approach- interest in cancer and the effects of atomic radiation on es, as well as classic homologous recombination-based mammals. These effects were investigated using mice, targeting in mouse embryonic stem cells (mESCs) that principally at the Oak Ridge National Laboratory in Oak served as the basis for the myriad knockout, knock-in, Ridge, Tennessee, and the MRC Radiobiological Research knockdown, conditional, and lineage-tracing mouse mod- Unit in Harwell, England. This research generated numer- els that now exist. More recently, nuclease-mediated ous mutant mouse strains, but because X-rays induce dou- technologies such as CRISPR/Cas have led to a second ex- ble-stranded breaks (DSBs) in DNA, the approach often plosion of techniques. These not only promise an easier, caused large deletions and chromosomal rearrangements more efficient means to traditional genetic ends, but are that affected multiple genes, making interpretation of also opening the door to ways of modifying the genome the mutant phenotypes difficult (Silver 1995). Radiation that were previously impractical or impossible with clas- has been supplanted by chemical mutagenesis with the sical techniques. DNA alkylating agent N-ethyl-N-nitrosourea (ENU), which is more mutagenic than X-rays (Russell et al. 1979) and more likely to result in point mutations (Popp Forward genetic screens et al. 1983). Because screening large numbers of mice in the G3 generation is so time- and labor-intensive, there Forward genetic screens involve germline mutagenesis are several modified approaches that allow for recovery and subsequent breeding of male mice to obtain a collec- of recessive mutations in the G2 generation. Specifically, tion of novel mutations. Offspring in the first generation modifier (enhancer or suppressor) and noncomplementa- (termed G1, the result of crossing a mutagenized mouse tion screens are performed by crossing G1 mutants with with a wild-type female) can be immediately screened an existing mutant strain and assaying for a modified phe- for dominant mutations giving rise to a phenotype of in- notype or failure to complement. This usually identifies terest. For recessive mutations, G1 males can be crossed new genes that genetically interact with the gene of inter- with a wild-type female and then any G2 females are est or novel alleles of the same gene, respectively. Dele- crossed back to their G1 father to generate homozygous tion screens, in which G1 males are crossed to females offspring in the third generation (G3). After identifying carrying a deletion of a chromosomal interval, facilitate mutant lines with the desired phenotype, the underlying rapid identification of mutations in this region. Finally, mutation is identified. This was traditionally achieved balancer screens, most famous from the world of fly genet- by time-consuming genetic mapping but can now be re- ics, can also be performed in mice even though the num- solved through genome sequencing, greatly accelerating ber of existing balancer chromosomes is limited (Zheng the overall process (Wang et al. 2015; Geister et al. et al. 1999). These involve crossing mutagenized males 2018). The advantage of genetic screens is that mutations to mice carrying a balancer chromosome, which has the are generated semi-randomly throughout the genome, so following features: one or a series of tandem chromosomal screens are unbiased with respect to the underlying muta- inversions that suppress recombination within the region, tion and can reveal unexpected genotype–phenotype con- a dominant visible trait such as coat color, and often a re- nections. One reason for this is that such screens can cessive lethal mutation (Zheng et al. 1999; Ye et al. 2016). identify many genes important for a given process that This combination of factors allows efficient breeding have not yet been investigated or linked to that process.

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