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The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) system is an adaptive immune system in bacteria and archaea that has recently been exploited for genome engineering. Mutant mice can be generated in one step through direct delivery of the CRISPR/Cas9 components into a mouse zygote. Although the technology is robust, delivery remains a bottleneck, as it involves manual injection of the components into the pronuclei or the cytoplasm of mouse zygotes, which is technically demanding and inherently low throughput. To overcome this limitation, we employed electroporation as a means to deliver the CRISPR/Cas9 components, including Cas9 messenger RNA, single-guide RNA, and donor oligonucleotide, into mouse zygotes and recovered live mice with targeted nonhomologous end joining and homology-directed repair mutations with high efficiency. Our results demonstrate that mice carrying CRISPR/Cas9-mediated targeted mutations can be obtained with high efficiency by zygote electroporation.

The first point of our body’s contact with tactile stimuli (innocuous and noxious) is the epidermis, the outermost layer of skin that is largely composed of keratinocytes. Here, we sought to define the role that keratinocytes play in touch sensation in vivo and ex vivo. We show that optogenetic inhibition of keratinocytes decreases behavioral and cellular mechanosensitivity. These processes are inherently mediated by ATP signaling, as demonstrated by complementary cutaneous ATP release and degradation experiments. Specific deletion of P2X4 receptors in sensory neurons markedly decreases behavioral and primary afferent mechanical sensitivity, thus positioning keratinocyte-released ATP to sensory neuron P2X4 signaling as a critical component of baseline mammalian tactile sensation. These experiments lay a vital foundation for subsequent studies into the dysfunctional signaling that occurs in cutaneous pain and itch disorders, and ultimately, the development of novel topical therapeutics for these conditions. The skin is the largest sensory organ of the body, and the first point of contact with the outside world. Whether it is being pinched or caressed, the skin’s sense of touch informs organisms about their surroundings and allows them to react appropriately. Nerve cells present in the skin capture information about touch and transmit it to the brain where it is decoded. However, there are many other types of cells in the skin besides nerve cells. The role that these other skin cells play in perceiving non-painful and painful touch is still unclear. Moehring et al. now report how the skin cells that form 95% of the most outer layer of the skin are involved in detecting touch. In mutant mice whose cells can be ‘switched off’ by a certain light, artificially deactivating these cells makes the animals less able to respond to tactile stimuli. Further experiments show that when pressure is applied onto the skin, the surface skin cells release a chemical messenger, which then binds specifically to the nerve cells. When the messaging molecule is experimentally destroyed or prevented from attaching to the nerve cell, the mice react less to non-painful and painful touch. This means the cells at the surface of the skin detect tactile signals from the environment and then communicate this information to the nerve cells, where it is taken to the brain. Disrupted communication between the cells in the outer layer of the skin and the nerve cells is found in painful and itchy skin conditions such as eczema and psoriasis. Knowing how these two types of cells normally work together may help with finding new pain and itch treatments for these skin disorders.

Future prospects continue to be strong for research using the rat as a model organism. New technology has enabled the proliferation of many new transgenic and knockout rat strains, the genomes of more than 40 rat strains have been sequenced, publications using the rat as a model continue to be produced at a steady rate, and discoveries of disease-associated genes and mechanisms from rat experiments abound, frequently with conservation of function between rats and humans. However, advances in genome technology have led to increasing insights into human disease directly from human genetic studies, pulling more and more researchers into the human genetics arena and placing funding for model organisms and their databases under threat. This, therefore, is a pivotal time for rat-based biomedical research – a timely moment to review progress and prospects – providing the inspiration for a new Special Collection focused on the impact of the model on translational science, launched in this issue of Disease Models & Mechanisms. What disease areas are most appropriate for research using rats? Why should the rat be favoured over other model organisms, and should the present levels of funding be continued? Which approaches should we expect to yield biologically and medically useful insights in the coming years? These are key issues that are addressed in the original Research Articles and reviews published in this Special Collection, and in this introductory Editorial. These exemplar articles serve as a landmark for the present status quo after a decade of major advances using the rat model and could help to guide the direction of rat research in the coming decade.

Measuring animal behavior in the context of experimental manipulation is critical for modeling, and understanding neuropsychiatric disease. Prepulse inhibition of the acoustic startle response (PPI) is a behavioral phenomenon studied extensively for this purpose, but the results of PPI studies are often inconsistent. As a result, the utility of this phenomenon remains uncertain. Here, we deconstruct the phenomenon of PPI and confirm several limitations of the methodology traditionally utilized to describe PPI, including that the underlying startle response has a non-Gaussian distribution, and that the traditional PPI metric changes with different stimuli. We then develop a novel model that reveals PPI to be a combination of the previously appreciated scaling of the startle response, as well as a scaling of sound processing. Using our model, we find no evidence for differences in PPI in a rat model of Fragile-X Syndrome (FXS) compared with wild-type controls. These results in the rat provide a reliable methodology that could be used to clarify inconsistent PPI results in mice and humans. In contrast, we find robust differences between wild-type male and female rats. Our model allows us to understand the nature of these differences, and we find that both the startle-scaling and sound-scaling components of PPI are a function of the baseline startle response. Males and females differ specifically in the startle-scaling, but not the sound-scaling, component of PPI. These findings establish a robust experimental and analytical approach that has the potential to provide a consistent biomarker of brain function.

Background A common feature of single-cell RNA-seq (scRNA-seq) data is that the number of cells in a cell cluster may vary widely, ranging from a few dozen to several thousand. It is not clear whether scRNA-seq data from a small number of cells allow robust identification of differentially expressed genes (DEGs) with various characteristics. Results We addressed this question by performing scRNA-seq and poly(A)-dependent bulk RNA-seq in comparable aliquots of human induced pluripotent stem cells-derived, purified vascular endothelial and smooth muscle cells. We found that scRNA-seq data needed to have 2,000 or more cells in a cluster to identify the majority of DEGs that would show modest differences in a bulk RNA-seq analysis. On the other hand, clusters with as few as 50–100 cells may be sufficient for identifying the majority of DEGs that would have extremely small p values or transcript abundance greater than a few hundred transcripts per million in a bulk RNA-seq analysis. Conclusion Findings of the current study provide a quantitative reference for designing studies that aim for identifying DEGs for specific cell clusters using scRNA-seq data and for interpreting results of such studies.

Recessive dystrophic epidermolysis bullosa is a rare genodermatosis caused by a mutation of the Col7a1 gene. The Col7a1 gene codes for collagen type VII protein, a major component of anchoring fibrils. Mutations of the Col7a1 gene can cause aberrant collagen type VII formation, causing an associated lack or absence of anchoring fibrils. This presents clinically as chronic blistering, scarring, and fibrosis, often leading to the development of cutaneous squamous cell carcinoma. Patients also experience persistent pain and pruritus. Pain management and supportive bandaging remain the primary treatment options. The pathology of recessive dystrophic epidermolysis bullosa was first described in the 1980s, and there has since been a multitude of encouraging treatment options developed. However, in vivo research has been hindered by inadequate models of the disease. The various mouse models in existence possess longevity and surface area constraints, or do not adequately model a normal human disease state. In this paper, we describe a novel rat model of recessive dystrophic epidermolysis bullosa that offers an alternative to previous murine models. An 8-base pair deletion was induced in the Col7a1 gene of Lewis rats, which was subsequently found to cause a premature stop codon downstream. Homozygous mutants presented with a fragile and chronically blistered phenotype postnatally. Further histological analysis revealed subepidermal clefting and the absence of anchoring fibrils. The generation of this novel model offers researchers an easily maintained organism that possesses a larger surface area for experimental topical and transfused therapies to be tested, which may provide great utility in the future study of this debilitating disease.

The toolbox of rat genetics currently lacks the ability to introduce site-directed, heritable mutations into the genome to create knockout animals. By using engineered zinc-finger nucleases (ZFNs) designed to target an integrated reporter and two endogenous rat genes, Immunoglobulin M (IgM) and Rab38, we demonstrate that a single injection of DNA or messenger RNA encoding ZFNs into the one-cell rat embryo leads to a high frequency of animals carrying 25 to 100% disruption at the target locus. These mutations are faithfully and efficiently transmitted through the germline. Our data demonstrate the feasibility of targeted gene disruption in multiple rat strains within 4 months time, paving the way to a humanized monoclonal antibody platform and additional human disease models.

Antithrombin III, encoded by SerpinC1, is a major anti-coagulation molecule in vivo and has anti-inflammatory effects. We found that patients with low antithrombin III activities presented a higher risk of developing acute kidney injury after cardiac surgery. To study this further, we generated SerpinC1 heterozygous knockout rats and followed the development of acute kidney injury in a model of modest renal ischemia/reperfusion injury. Renal injury, assessed by serum creatinine and renal tubular injury scores after 24 h of reperfusion, was significantly exacerbated in SerpinC1+/− rats compared to wild-type littermates. Concomitantly, renal oxidative stress, tubular apoptosis, and macrophage infiltration following this injury were significantly aggravated in SerpinC1+/− rats. However, significant thrombosis was not found in the kidneys of any group of rats. Antithrombin III is reported to stimulate the production of prostaglandin I2, a known regulator of renal cortical blood flow, in addition to having anti-inflammatory effects and to protect against renal failure. Prostaglandin F1α, an assayable metabolite of prostaglandin I2, was increased in the kidneys of the wild-type rats at 3 h after reperfusion. The increase of prostaglandin F1α was significantly blunted in SerpinC1+/− rats, which preceded increased tubular injury and oxidative stress. Thus, our study found a novel role of SerpinC1 insufficiency in increasing the severity of renal ischemia/reperfusion injury.

The heptapeptide angiotensin-(1-7) (Ang-(1-7)) is protective in the cardiovascular system through its induction of vasodilator production and angiogenesis. Despite acting antagonistically to the effects of elevated, pathophysiological levels of angiotensin II (AngII), recent evidence has identified convergent and beneficial effects of low levels of both Ang-(1-7) and AngII. Previous work identified the AngII receptor type I (AT1R) as a component of the protein complex formed when Ang-(1-7) binds its receptor, Mas1. Importantly, pharmacological blockade of AT1R did not alter the effects of Ang-(1-7). Here, we use a novel mutation of AT1RA in the Dahl salt-sensitive (SS) rat to test the hypothesis that interaction between Mas1 and AT1R contributes to proangiogenic Ang-(1-7) signaling. In a model of hind limb angiogenesis induced by electrical stimulation, we find that the restoration of skeletal muscle angiogenesis in SS rats by Ang-(1-7) infusion is impaired in AT1RA knockout rats. Enhancement of endothelial cell (EC) tube formation capacity by Ang-(1-7) is similarly blunted in AT1RA mutant ECs. Transcriptional changes elicited by Ang-(1-7) in SS rat ECs are altered in AT1RA mutant ECs, and tandem mass spectrometry-based proteomics demonstrate that the protein complex formed upon binding of Ang-(1-7) to Mas1 is altered in AT1RA mutant ECs. Together, these data support the hypothesis that interaction between AT1R and Mas1 contributes to proangiogenic Ang-(1-7) signaling.

Background SHC1 proteins (also called SHCA) exist in three functionally distinct isoforms (p46SHC, p52SHC, and p66SHC) that serve as intracellular adaptors for several key signaling pathways in breast cancer. Despite the broad evidence implicating SHC1 gene products as a central mediator of breast cancer, testing the isoform-specific roles of SHC1 proteins have been inaccessible due to the lack of isoform-specific inhibitors or gene knockout models. Methods Here, we addressed this issue by generating the first isoform-specific gene knockout models for p52SHC and p66SHC, using germline gene editing in the salt-sensitive rat strain. Compared with the wild-type (WT) rats, we found that genetic ablation of the p52SHC isoform significantly attenuated mammary tumor formation, whereas the p66SHC knockout had no effect. Rats were dosed with 7,12-dimethylbenz(a)anthracene (DMBA) by oral gavage to induce mammary tumors, and progression of tumor development was followed for 15 weeks. At 15 weeks, tumors were excised and analyzed by RNA-seq to determine differences between tumors lacking p66SHC or p52SHC. Results Compared with the wild-type (WT) rats, we found that genetic ablation of the p52SHC isoform significantly attenuated mammary tumor formation, whereas the p66SHC knockout had no effect. These data, combined with p52SHC being the predominant isoform that is upregulated in human and rat tumors, provide the first evidence that p52SHC is the oncogenic isoform of Shc1 gene products in breast cancer. Compared with WT tumors, 893 differentially expressed (DE; FDR < 0.05) genes were detected in p52SHC KO tumors compared with only 18 DE genes in the p66SHC KO tumors, further highlighting that p52SHC is the relevant SHC1 isoform in breast cancer. Finally, gene network analysis revealed that p52SHC KO disrupted multiple key pathways that have been previously implicated in breast cancer initiation and progression, including ESR1 and mTORC2/RICTOR. Conclusion Collectively, these data demonstrate the p52SHC isoform is the key driver of DMBA-induced breast cancer while the expression of p66SHC and p46SHC are not enough to compensate.