Michael V. Wiles

Mouse Estrous Cycle Identification Tool and Images

The efficiency of producing timed pregnant or pseudopregnant mice can be increased by identifying those in proestrus or estrus. Visual observation of the vagina is the quickest method, requires no special equipment, and is best used when only proestrus or estrus stages need to be identified. Strain to strain differences, especially in coat color can make it difficult to determine the stage of the estrous cycle accurately by visual observation. Presented here are a series of images of the vaginal opening at each stage of the estrous cycle for 3 mouse strains of different coat colors: black (C57BL/6J), agouti (CByB6F1/J) and albino (BALB/cByJ). When all 4 stages (proestrus, estrus, metestrus, and diestrus) need to be identified, vaginal cytology is regarded as the most accurate method. An identification tool is presented to aid the user in determining the stage of estrous when using vaginal cytology. These images and descriptions are an excellent resource for learning how to determine the stage of the estrous cycle by visual observation or vaginal cytology.

Conserving, distributing and managing genetically modified mouse lines by sperm cryopreservation.

Background Sperm from C57BL/6 mice are difficult to cryopreserve and recover. Yet, the majority of genetically modified (GM) lines are maintained on this genetic background. Methodology/Principal Findings Reported here is the development of an easily implemented method that consistently yields fertilization rates of 70±5% with this strain. This six-fold increase is achieved by collecting sperm from the vas deferens and epididymis into a cryoprotective medium of 18% raffinose (w/v), 3% skim milk (w/v) and 477 µM monothioglycerol. The sperm suspension is loaded into 0.25 mL French straws and cooled at 37±1°C/min before being plunged and then stored in LN2. Subsequent to storage, the sperm are warmed at 2,232±162°C/min and incubated in in vitro fertilization media for an hour prior to the addition of oocyte cumulus masses from superovulated females. Sperm from 735 GM mouse lines on 12 common genetic backgrounds including C57BL/6J, BALB/cJ, 129S1/SvImJ, FVB/NJ and NOD/ShiLtJ were cryopreserved and recovered. C57BL/6J and BALB/cByJ fertilization rates, using frozen sperm, were slightly reduced compared to rates involving fresh sperm; fertilization rates using fresh or frozen sperm were equivalent in all other lines. Developmental capacity of embryos produced using cryopreserved sperm was equivalent, or superior to, cryopreserved IVF-derived embryos. Conclusions/Significance Combined, these results demonstrate the broad applicability of our approach as an economical and efficient option for archiving and distributing mice.

A radiation hybrid transcript map of the mouse genome.

Expressed-sequence tag (EST) maps are an adjunct to sequence-based analytical methods of gene detection and localization for those species for which such data are available, and provide anchors for high-density homology and orthology mapping in species for which large-scale sequencing has yet to be done. Species for which radiation hybrid–based transcript maps have been established include human, rat, mouse, dog, cat and zebrafish. We have established a comprehensive first-generation–placement radiation hybrid map of the mouse consisting of 5,904 mapped markers (3,993 ESTs and 1,911 sequence-tagged sites (STSs)). The mapped ESTs, which often originate from small-EST clusters, are enriched for genes expressed during early mouse embryogenesis and are probably different from those localized in humans. We have confirmed by in situ hybridization that even singleton ESTs, which are usually not retained for mapping studies, may represent bona fide transcribed sequences. Our studies on mouse chromosomes 12 and 14 orthologous to human chromosome 14 show the power of our radiation hybrid map as a predictive tool for orthology mapping in humans.

Albumin-deficient mouse models for studying metabolism of human albumin and pharmacokinetics of albumin-based drugs.

Serum albumin is the major determinant of blood colloidal osmotic pressure acting as a depot and distributor of compounds including drugs. In humans, serum albumin exhibits an unusually long half-life mainly due to protection from catabolism by neonatal Fc receptor (FcRn)-mediated recycling. These properties make albumin an attractive courier of therapeutically-active compounds. However, pharmaceutical research and development of albumin-based therapeutics has been hampered by the lack of appropriate preclinical animal models. To overcome this, we developed and describe the first mouse with a genetic deficiency in albumin and its incorporation into an existing humanized FcRn mouse model, B6.Cg-Fcgrttm1Dcr Tg(FCGRT)32Dcr/DcrJ (Tg32). Albumin-deficient strains (Alb-/-) were created by TALEN-mediated disruption of the albumin (Alb) gene directly in fertilized oocytes derived from Tg32 mice and its non-transgenic background control, C57BL/6J (B6). The resulting Alb-/- strains are analbuminemic but healthy. Intravenous administration of human albumin to Tg32-Alb-/- mFcRn-/- hFcRnTg/Tg) mice results in a remarkably extended human albumin serum half-life of ∼24 days, comparable to that found in humans, and in contrast to half-lives of 2.6–5.8 d observed in B6, B6-Alb-/- and Tg32 strains. This striking increase can be explained by the absence of competing endogenous mouse albumin and the presence of an active human FcRn. These novel albumin-deficient models provide unique tools for investigating the biology and pathobiology of serum albumin and are a more appropriate rodent surrogates for evaluating human serum albumin pharmacokinetics and albumin-based compounds.

Simple, Efficient CRISPR-Cas9-Mediated Gene Editing in Mice: Strategies and Methods

Genetic modification of almost any species is now possible using approaches based on targeted nucleases. These novel tools now bypass previous limited species windows, allowing precision nucleotide modification of the genome at high efficiency, rapidly and economically. Here we focus on the modification of the mouse genome; the mouse, with its short generation time and comparatively low maintenance/production costs is the perfect mammal with which to probe the genome to understand its functions and complexities. Further, using targeted nucleases combined with homologous recombination, it is now possible to precisely tailor the genome, creating models of human diseases and conditions directly and efficiently in zygotes derived from any mouse strain. Combined these approaches make it possible to sequentially and progressively refine mouse models to better reflect human disease, test and develop therapeutics. Here, we briefly review the strategies involved in designing targeted nucleases (sgRNAs) providing solutions and outlining in detail the practical processes involved in precision targeting and modification of the mouse genome and the establishing of new precision genetically modified mouse lines.

The Sophisticated Mouse: Protecting a Precious Reagent

Definable, genetically and environmentally, the humble mouse has become a reagent with which to probe the human condition. The information thus gained is leading to a greater understanding of inter-individual variation in drug responses and disease processes and is forming the basis for personalized medicine. Inbred mice are the tool of choice as each strain is essentially clonal in nature creating a defined, uniform setting where the effects of genetic background and modifications can be evaluated coherently. However, the creation and characterization of novel mouse strains remain expensive and time consuming. Further, the continual maintenance of these valuable animals as live colonies is financially draining and carries continual potential risks, including disastrous loss due to fire, flood, disease, etc. There are also other more insidious disasters including genetic contamination and genetic drift, either of which can go undiscovered until their effects ruin experiments. With this in mind, we strongly recommend that all mouse strains be cryopreserved as a matter of standard mouse management. Cryopreservation is a powerful colony management tool, assuring strains are available upon demand, for example, for regulatory requirements, re-initiation of projects, collaborations, re-evaluation of data etc. However, it is essential that any cryopreservation approach be cost-effective for both strain closure and strain recovery. In this chapter, we describe the variables which can afflict an inbred mouses genetic background (and hence phenotype), options to consider for strain archiving, and describe how to economically store and recover strains by sperm cryopreservation.

Genetically Engineered Humanized Mouse Models for Preclinical Antibody Studies

The use of genetic engineering has vastly improved our capabilities to create animal models relevant in preclinical research. With the recent advances in gene-editing technologies, it is now possible to very rapidly create highly tunable mouse models as needs arise. Here, we provide an overview of genetic engineering methods, as well as the development of humanized neonatal Fc receptor (FcRn) models and their use for monoclonal antibody in vivo studies.

The Perfect Host: A Mouse Host Embryo Facilitating More Efficient Germ Line Transmission of Genetically Modified Embryonic Stem Cells

There is a continual need to improve efficiency in creating precise genetic modifications in mice using embryonic stem cells (ESCs). We describe a novel approach resulting in 100% germline transmission from competent injected ESCs. We developed an F1 mouse host embryo (Perfect Host, PH) that selectively ablates its own germ cells via tissue-specific induction of diphtheria toxin. This approach allows competent microinjected ESCs to fully dominate the germline, eliminating competition for this critical niche in the developing and adult animal. This is in contrast to conventional methods, where competition from host germ cells results in offspring derived from host cells and ESCs, necessitating extensive breeding of chimeras and genotyping to identify germline. The germline transmission process is also complicated by variability in the actual number of ESCs that colonize the germline niche and the proportion that are germline competent. To validate the PH approach we used ESC lines derived from 129 F1, BALB/cByJ, and BTBR backgrounds as well as an iPS line. Resulting chimeric males produced 194 offspring, all paternally derived from the introduced stem cells, with no offspring being derived from the host genome. We further tested this approach using eleven genetically modified C57BL/6N ESC lines (International Knockout Mouse Consortium). ESC germline transmission was observed in 9/11 (82%) lines using PH blastocysts, compared to 6/11 (55%) when conventional host blastocysts were used. Furthermore, less than 35% (83/240) of mice born in the first litters from conventional chimeras were confirmed to be of ESC-origin. By comparison, 100% (137/137) of the first litter offspring of PH chimeras were confirmed as ESC-derived. Together, these data demonstrate that the PH approach increases the probability of germline transmission and speeds the generation of ESC derived animals from chimeras. Collectively, this approach reduces the time and costs inherent in the production of genetically modified animals.

Mouse Models for Drug Discovery

There is growing concern about the poor quality and lack of repeatability of many pre-clinical experiments involving laboratory animals. According to one estimate as much as

A Humanized Mouse Model to Study Human Albumin and Albumin Conjugates Pharmacokinetics

28 billion is wasted annually in the USA alone in such studies. A decade ago the FDA’s “Critical path” white paper noted that “The traditional tools used to assess product safety—animal toxicology and outcomes from human studies—have changed little over many decades and have largely not benefited from recent gains in scientific knowledge. The inability to better assess and predict product safety leads to failures during clinical development and, occasionally, after marketing.” Repeat-dose 28-days and 90-days toxicity tests in rodents have been widely used as part of a strategy to assess the safety of drugs and chemicals but their repeatability and power to detect adverse effects have not been formally evaluated. The guidelines (OECD TG 407 and 408) for these tests specify the dose levels and number of animals per dose but do not specify the strain of animals which should be used. In practice, almost all the tests are done using genetically undefi ned “albino” rats or mice in which the genetic variation, a major cause of inter-individual and strain variability, is unknown and uncontrolled. This chapter suggests that a better strategy would be to use small numbers of animals of several genetically defi ned strains of mice or rats instead of the undefi ned animals used at present. Inbred strains are more stable providing more repeatable data than outbred stocks. Importantly their greater phenotypic uniformity should lead to more powerful and repeatable tests. Any observed strain differences would indicate genetic variation in response to the test substance, providing key data. We suggest that the FDA and other regulators and funding organizations should support research to evaluate this alternative.