ADAR1 averts fatal type I interferon induction by ZBP1.

Mutations of the ADAR1 gene encoding an RNA deaminase cause severe diseases associated with chronic activation of type I interferon (IFN) responses, including Aicardi-Goutières syndrome and bilateral striatal necrosis1-3. The IFN-inducible p150 isoform of ADAR1 contains a Zα domain that recognizes RNA with an alternative left-handed double-helix structure, termed Z-RNA4,5. Hemizygous ADAR1 mutations in the Zα domain cause type I IFN-mediated pathologies in humans2,3 and mice6-8; however, it remains unclear how the interaction of ADAR1 with Z-RNA prevents IFN activation. Here we show that Z-DNA-binding protein 1 (ZBP1), the only other protein in mammals known to harbour Zα domains9, promotes type I IFN activation and fatal pathology in mice with impaired ADAR1 function. ZBP1 deficiency or mutation of its Zα domains reduced the expression of IFN-stimulated genes and largely prevented early postnatal lethality in mice with hemizygous expression of ADAR1 with mutated Zα domain (Adar1mZα/- mice). Adar1mZα/- mice showed upregulation and impaired editing of endogenous retroelement-derived complementary RNA reads, which represent a likely source of Z-RNAs activating ZBP1. Notably, ZBP1 promoted IFN activation and severe pathology in Adar1mZα/- mice in a manner independent of RIPK1, RIPK3, MLKL-mediated necroptosis and caspase-8-dependent apoptosis, suggesting a novel mechanism of action. Thus, ADAR1 prevents endogenous Z-RNA-dependent activation of pathogenic type I IFN responses by ZBP1, suggesting that ZBP1 could contribute to type I interferonopathies caused by ADAR1 mutations.

SARM1 deletion delays cerebellar but not spinal cord degeneration in an enhanced mouse model of SPG7 deficiency.

Hereditary spastic paraplegia is a neurological condition characterized by predominant axonal degeneration in long spinal tracts, leading to weakness and spasticity in the lower limbs. The nicotinamide adenine dinucleotide (NAD+)-consuming enzyme SARM1 has emerged as a key executioner of axonal degeneration upon nerve transection and in some neuropathies. An increase in the nicotinamide mononucleotide/NAD+ ratio activates SARM1, causing catastrophic NAD+ depletion and axonal degeneration. However, the role of SARM1 in the pathogenesis of hereditary spastic paraplegia has not been investigated. Here, we report an enhanced mouse model for hereditary spastic paraplegia caused by mutations in SPG7. The eSpg7 knockout mouse carries a deletion in both Spg7 and Afg3l1, a redundant homologue expressed in mice but not in humans. The eSpg7 knockout mice recapitulate the phenotypic features of human patients, showing progressive symptoms of spastic-ataxia and degeneration of axons in the spinal cord as well as the cerebellum. We show that the lack of SPG7 rewires the mitochondrial proteome in both tissues, leading to an early onset decrease in mito-ribosomal subunits and a remodelling of mitochondrial solute carriers and transporters. To interrogate mechanisms leading to axonal degeneration in this mouse model, we explored the involvement of SARM1. Deletion of SARM1 delays the appearance of ataxic signs, rescues mitochondrial swelling and axonal degeneration of cerebellar granule cells and dampens neuroinflammation in the cerebellum. The loss of SARM1 also prevents endoplasmic reticulum abnormalities in long spinal cord axons, but does not halt the degeneration of these axons. Our data thus reveal a neuron-specific interplay between SARM1 and mitochondrial dysfunction caused by lack of SPG7 in hereditary spastic paraplegia.

Inflammatory reactions and severe neutropenia in mice lacking the transcriptional repressor Gfi1.

The transcriptional repressor Gfi1 is a nuclear zinc-finger protein expressed in T-cell precursors in the thymus and in activated mature T lymphocytes. Previous experiments have shown that Gfi1 is involved in T-cell lymphomagenesis and in the development of T-cell progenitors. Here we show that Gfi1 is also expressed outside the lymphoid system in granulocytes and activated macrophages, cells that mediate innate immunity (that is, non-specific immunity). We have generated Gfi1-deficient mice (Gfi1-/-) and show that these animals are severely neutropenic and accumulate immature monocytic cells in blood and bone marrow. Their myeloid precursor cells are unable to differentiate into granulocytes upon stimulation with granulocyte colony-stimulating factor (G-CSF) but can develop into mature macrophages. We found that Gfi1-/- macrophages produce enhanced levels of inflammatory cytokines, such as tumor necrosis factor (TNF), interleukin-10 (IL-10) and IL-1beta, when stimulated with bacterial lipopolysaccharide (LPS) and that Gfi1-/- mice succumb to low doses of this endotoxin that are tolerated by wildtype mice. We conclude that Gfi1 influences the differentiation of myeloid precursors into granulocytes or monocytes and acts in limiting the inflammatory immune response.

Practical Application of the 3Rs in Rodent Transgenesis.

The principles of the 3Rs (replace, reduce, refine), as originally published by Russell and Burch, are internationally acclaimed guidelines for meeting ethical and welfare standards in animal experimentation. Genome manipulation is a standard technique in biomedical research and beyond. The goal of this chapter is to give practical advice on the implementation of the 3Rs in laboratories generating genetically modified rodents. We cover 3R aspects from the planning phase through operations of the transgenic unit to the final genome-manipulated animals. The focus of our chapter is on an easy-to-use, concise protocol that is close to a checklist. While we focus on mice, the proposed methodological concepts can be easily adapted for the manipulation of other sentient animals.

Successful use of HTF as a basal fertilization medium during SEcuRe mouse in vitro fertilization.

OBJECTIVE: The ever-increasing number of genetically engineered mouse models highlights the need for efficient archiving and distribution of these lines. Sperm cryopreservation has become the preferred technique for the majority of these models due to its low requirement of costs, time, and experimental animals. Yet, current in vitro fertilization (IVF) protocols either exhibit decreased fertilization efficiency for the most popular C57BL/6 strain, as recently demonstrated by us, or require costly and difficult-to-prepare media, respectively. As a result, we previously developed SEcuRe, a modified IVF protocol with low costs and high fertilization efficiency. The popular basal fertilization medium, Cook's® proprietary "Research vitro fert" (RVF), used in this protocol has recently been discontinued. As a result, the application of the SEcuRe approach and other IVF protocols employing this medium has been severely limited. RESULTS: Here we show that human tubal fluid (HTF), a popular and widely available medium with a known composition, can be used as a basal fertilization medium instead of RVF. Comparison of RVF and HTF during 58 independent SEcuRe IVFs with cryopreserved C57BL/6 sperm revealed equal fertilization and live birth rates. In addition, we demonstrate that HTF has a substantially extended shelf-life by utilizing commercial HTF that was six months past its expiration date, yet did not affect fertilization during IVF or subsequent embryo development. This finding not only increases the economic value of our modified method, but also validates it once more. Our results demonstrate that common, shelf-life extended HTF can be used in SEcuRe IVF in place of now-discontinued RVF medium and ensure the applicability of the method, which we since termed SEcuRe 2.0. Our modified SEcuRe 2.0 strategy will assist researchers to efficiently archive and distribute genetically engineered mouse models in a cost-effective, easily adaptable, and 3R-compliant manner with minimal animal use.

Distinct Genetically Determined Origins of Myd88/BCL2-Driven Aggressive Lymphoma Rationalize Targeted Therapeutic Intervention Strategies.

Genomic profiling revealed the identity of at least 5 subtypes of diffuse large B-cell lymphoma (DLBCL), including the MCD/C5 cluster characterized by aberrations in MYD88, BCL2, PRDM1, and/or SPIB. We generated mouse models harboring B cell-specific Prdm1 or Spib aberrations on the background of oncogenic Myd88 and Bcl2 lesions. We deployed whole-exome sequencing, transcriptome, flow-cytometry, and mass cytometry analyses to demonstrate that Prdm1- or Spib-altered lymphomas display molecular features consistent with prememory B cells and light-zone B cells, whereas lymphomas lacking these alterations were enriched for late light-zone and plasmablast-associated gene sets. Consistent with the phenotypic evidence for increased B cell receptor signaling activity in Prdm1-altered lymphomas, we demonstrate that combined BTK/BCL2 inhibition displays therapeutic activity in mice and in five of six relapsed/refractory DLBCL patients. Moreover, Prdm1-altered lymphomas were immunogenic upon transplantation into immuno-competent hosts, displayed an actionable PD-L1 surface expression, and were sensitive to antimurine-CD19-CAR-T cell therapy, in vivo. SIGNIFICANCE: Relapsed/refractory DLBCL remains a major medical challenge, and most of these patients succumb to their disease. Here, we generated mouse models, faithfully recapitulating the biology of MYD88-driven human DLBCL. These models revealed robust preclinical activity of combined BTK/BCL2 inhibition. We confirmed activity of this regimen in pretreated non-GCB-DLBCL patients. See related commentary by Leveille et al., p. 8. This article is highlighted in the In This Issue feature, p. 1.

History of genome editing: From meganucleases to CRISPR.

CRISPR-mediated genome editing has undoubtedly revolutionized genetic engineering of animals. With the ability for virtually unlimited modification of almost any genome it is easy to forget which amazing discoveries paved the way for this ground-breaking technology. Here, we summarize the history of genome editing platforms, starting from enhanced integration of foreign DNA by meganuclease-mediated double-strand breaks to CRISPR/Cas9, the leading technology to date, and its re-engineered variants.

3R measures in facilities for the production of genetically modified rodents.

Sociocultural changes in the human-animal relationship have led to increasing demands for animal welfare in biomedical research. The 3R concept is the basis for bringing this demand into practice: Replace animal experiments with alternatives where possible, Reduce the number of animals used to a scientifically justified minimum and Refine the procedure to minimize animal harm. The generation of gene-modified sentient animals such as mice and rats involves many steps that include various forms of manipulation. So far, no coherent analysis of the application of the 3Rs to gene manipulation has been performed. Here we provide guidelines from the Committee on Genetics and Breeding of Laboratory Animals of the German Society for Laboratory Animal Science to implement the 3Rs in every step during the generation of genetically modified animals. We provide recommendations for applying the 3Rs as well as success/intervention parameters for each step of the process, from experiment planning to choice of technology, harm-benefit analysis, husbandry conditions, management of genetically modified lines and actual procedures. We also discuss future challenges for animal welfare in the context of developing technologies. Taken together, we expect that our comprehensive analysis and our recommendations for the appropriate implementation of the 3Rs to technologies for genetic modifications of rodents will benefit scientists from a wide range of disciplines and will help to improve the welfare of a large number of laboratory animals worldwide.

A novel panel of mouse models to evaluate the role of human pregnane X receptor and constitutive androstane receptor in drug response.

The pregnane X receptor (PXR) and the constitutive androstane receptor (CAR) are closely related orphan nuclear hormone receptors that play a critical role as xenobiotic sensors in mammals. Both receptors regulate the expression of genes involved in the biotransformation of chemicals in a ligand-dependent manner. As the ligand specificity of PXR and CAR have diverged between species, the prediction of in vivo PXR and CAR interactions with a drug are difficult to extrapolate from animals to humans. We report the development of what we believe are novel PXR- and CAR-humanized mice, generated using a knockin strategy, and Pxr- and Car-KO mice as well as a panel of mice including all possible combinations of these genetic alterations. The expression of human CAR and PXR was in the predicted tissues at physiological levels, and splice variants of both human receptors were expressed. The panel of mice will allow the dissection of the crosstalk between PXR and CAR in the response to different drugs. To demonstrate the utility of this panel of mice, we used the mice to show that the in vivo induction of Cyp3a11 and Cyp2b10 by phenobarbital was only mediated by CAR, although this compound is described as a PXR and CAR activator in vitro. This panel of mouse models is a useful tool to evaluate the roles of CAR and PXR in drug bioavailability, toxicity, and efficacy in humans.

A simple and economic protocol for efficient in vitro fertilization using cryopreserved mouse sperm.

The advent of genome editing tools like CRISPR/Cas has substantially increased the number of genetically engineered mouse models in recent years. In support of refinement and reduction, sperm cryopreservation is advantageous compared to embryo freezing for archiving and distribution of such mouse models. The in vitro fertilization using cryopreserved sperm from the most widely used C57BL/6 strain has become highly efficient in recent years due to several improvements of the procedure. However, purchase of the necessary media for routine application of the current protocol poses a constant burden on budgetary constraints. In-house media preparation, instead, is complex and requires quality control of each batch. Here, we describe a cost-effective and easily adaptable approach for in vitro fertilization using cryopreserved C57BL/6 sperm. This is mainly achieved by modification of an affordable commercial fertilization medium and a step-by-step description of all other necessary reagents. Large-scale comparison of fertilization rates from independent lines of genetically engineered C57BL/6 mice upon cryopreservation and in vitro fertilization with our approach demonstrated equal or significantly superior fertilization rates to current protocols. Our novel SEcuRe (Simple Economical set-up for Rederivation) method provides an affordable, easily adaptable and harmonized protocol for highly efficient rederivation using cryopreserved C57BL/6 sperm for a broad application of colony management in the sense of the 3Rs.

A MAFG-lncRNA axis links systemic nutrient abundance to hepatic glucose metabolism.

Obesity and type 2 diabetes mellitus are global emergencies and long noncoding RNAs (lncRNAs) are regulatory transcripts with elusive functions in metabolism. Here we show that a high fraction of lncRNAs, but not protein-coding mRNAs, are repressed during diet-induced obesity (DIO) and refeeding, whilst nutrient deprivation induced lncRNAs in mouse liver. Similarly, lncRNAs are lost in diabetic humans. LncRNA promoter analyses, global cistrome and gain-of-function analyses confirm that increased MAFG signaling during DIO curbs lncRNA expression. Silencing Mafg in mouse hepatocytes and obese mice elicits a fasting-like gene expression profile, improves glucose metabolism, de-represses lncRNAs and impairs mammalian target of rapamycin (mTOR) activation. We find that obesity-repressed LincIRS2 is controlled by MAFG and observe that genetic and RNAi-mediated LincIRS2 loss causes elevated blood glucose, insulin resistance and aberrant glucose output in lean mice. Taken together, we identify a MAFG-lncRNA axis controlling hepatic glucose metabolism in health and metabolic disease.

ES cell line establishment.

A method is described to establish mouse embryonic stem cell (ESC) lines from hybrid and inbred strains of mice. Attention is paid not only to the methodology for isolation and culture but also to the validation of freshly derived lines, in order to be maintained for prolonged time without significant differentiation or karyotype instability, and to provide reproducible germline transmission in chimaeric mice.

An optimized electroporation approach for efficient CRISPR/Cas9 genome editing in murine zygotes.

Electroporation of zygotes represents a rapid alternative to the elaborate pronuclear injection procedure for CRISPR/Cas9-mediated genome editing in mice. However, current protocols for electroporation either require the investment in specialized electroporators or corrosive pre-treatment of zygotes which compromises embryo viability. Here, we describe an easily adaptable approach for the introduction of specific mutations in C57BL/6 mice by electroporation of intact zygotes using a common electroporator with synthetic CRISPR/Cas9 components and minimal technical requirement. Direct comparison to conventional pronuclear injection demonstrates significantly reduced physical damage and thus improved embryo development with successful genome editing in up to 100% of living offspring. Hence, our novel approach for Easy Electroporation of Zygotes (EEZy) allows highly efficient generation of CRISPR/Cas9 transgenic mice while reducing the numbers of animals required.

Hybrid embryonic stem cell-derived tetraploid mice show apparently normal morphological, physiological, and neurological characteristics.

ES cell-tetraploid (ES) mice are completely derived from embryonic stem cells and can be obtained at high efficiency upon injection of hybrid ES cells into tetraploid blastocysts. This method allows the immediate generation of targeted mouse mutants from genetically modified ES cell clones, in contrast to the standard protocol, which involves the production of chimeras and several breeding steps. To provide a baseline for the analysis of ES mouse mutants, we performed a phenotypic characterization of wild-type B6129S6F(1) ES mice in relation to controls of the same age, sex, and genotype raised from normal matings. The comparison of 90 morphological, physiological, and behavioral parameters revealed elevated body weight and hematocrit as the only major difference of ES mice, which exhibited an otherwise normal phenotype. We further demonstrate that ES mouse mutants can be produced from mutant hybrid ES cells and analyzed within a period of only 4 months. Thus, ES mouse technology is a valid research tool for rapidly elucidating gene function in vivo.

Male and female mice derived from the same embryonic stem cell clone by tetraploid embryo complementation.

We have devised a general strategy for producing female mice from 39,X0 embryonic stem (ES) cells derived from male cell lines carrying a targeted mutation of interest. We show that the Y chromosome is lost in 2% of subclones from 40,XY ES cell lines, making the identification of targeted 39,X0 subclones a routine procedure. After gene targeting, male and female mice carrying the mutation can be generated by tetraploid embryo complementation from the 40,XY ES cell line and its 39,X0 derivatives. A single intercross then produces homozygous mutant offspring. Because this strategy avoids outcrossing and therefore segregation of mutant alleles introduced into the ES cells, the time and expense required for production of experimental mutant animals from a targeted ES cell clone are substantially reduced. Our data also indicate that ES cells have inherently unstable karyotypes, but this instability does not interfere with production of adult ES cell tetraploid mice.

Rapid generation of inducible mouse mutants.

We have generated an optimized inducible recombination system for conditional gene targeting based on a Cre recombinase-steroid receptor fusion. This configuration allows efficient Cre-mediated recombination in most organs of the mouse upon induction, without detectable background activity. An ES cell line, was established that carries the inducible recombinase and a loxP-flanked lacZ reporter gene. Out of this line, completely ES cell-derived mice were efficiently produced through tetraploid blastocyst complementation, without the requirement of mouse breeding. Our findings provide a new concept allowing the generation of inducible mouse mutants within 6 months, as compared to 14 months using the current protocol.

Manipulating the Prion Protein Gene Sequence and Expression Levels with CRISPR/Cas9.

The mammalian prion protein (PrP, encoded by Prnp) is most infamous for its central role in prion diseases, invariably fatal neurodegenerative diseases affecting humans, food animals, and animals in the wild. However, PrP is also hypothesized to be an important receptor for toxic protein conformers in Alzheimer's disease, and is associated with other clinically relevant processes such as cancer and stroke. Thus, key insights into important clinical areas, as well as into understanding PrP functions in normal physiology, can be obtained from studying transgenic mouse models and cell culture systems. However, the Prnp locus is difficult to manipulate by homologous recombination, making modifications of the endogenous locus rarely attempted. Fortunately in recent years genome engineering technologies, like TALENs or CRISPR/Cas9 (CC9), have brought exceptional new possibilities for manipulating Prnp. Herein, we present our observations made during systematic experiments with the CC9 system targeting the endogenous mouse Prnp locus, to either modify sequences or to boost PrP expression using CC9-based synergistic activation mediators (SAMs). It is our hope that this information will aid and encourage researchers to implement gene-targeting techniques into their research program.

C57BL/6N albino/agouti mutant mice as embryo donors for efficient germline transmission of C57BL/6 ES cells.

We generated C57BL/6NTac mice carrying a tyrosinase loss-of function mutation and a reversion of the nonagouti locus to agouti. This strain has a high superovulation response, allows visual detection of chimeric coat color contribution of C57BL/6 ES-cells and provides a simplified breeding format that generates black G1 offspring of pure inbred C57BL/6 background in one step, providing the ideal host for genetically manipulated C57BL/6 ES cells.

GPR30 does not mediate estrogenic responses in reproductive organs in mice.

The G protein-coupled receptor Gpr30 (Gper) was recently claimed to bind to estradiol and to activate cytoplasmic signal transduction pathways in response to estradiol. However, there are conflicting data regarding the role of Gpr30 as an estrogen receptor (ER): several laboratories were unable to demonstrate estradiol binding to GPR30 or estradiol-activated signal transduction in Gpr30-expressing cells. To clarify the potential role of Gpr30 as an ER, we generated Gpr30-deficient mice. Although Gpr30 was expressed in all reproductive organs, histopathological analysis did not reveal any abnormalities in these organs in Gpr30-deficient mice. Mutant male and female mice were as fertile as their wild-type littermates, indicating normal function of the hypothalamic-pituitary-gonadal axis. Moreover, we analyzed estrogenic responses in two major estradiol target organs, the uterus and the mammary gland. For that purpose, we examined different readout paradigms such as morphological measures, cellular proliferation, and target gene expression. Our data demonstrate that in vivo Gpr30 is dispensable for the mediation of estradiol effects in reproductive organs. These results are in clear contrast to the phenotype of mice lacking the classic ER alpha (Esr1) or aromatase (Cyp19a1). We conclude that the perception of Gpr30 (based on homology related to peptide receptors) as an ER might be premature and has to be reconsidered.

Epididymis seleno-independent glutathione peroxidase 5 maintains sperm DNA integrity in mice.

The mammalian epididymis provides sperm with an environment that promotes their maturation and protects them from external stresses. For example, it harbors an array of antioxidants, including non-conventional glutathione peroxidase 5 (GPX5), to protect them from oxidative stress. To explore the role of GPX5 in the epididymis, we generated mice that lack epididymal expression of the enzyme. Histological analyses of Gpx5-/- epididymides and sperm cells revealed no obvious defects. Furthermore, there were no apparent differences in the fertilization rate of sexually mature Gpx5-/- male mice compared with WT male mice. However, a higher incidence of miscarriages and developmental defects were observed when WT female mice were mated with Gpx5-deficient males over 1 year old compared with WT males of the same age. Flow cytometric analysis of spermatozoa recovered from Gpx5-null and WT male mice revealed that sperm DNA compaction was substantially lower in the cauda epididymides of Gpx5-null animals and that they suffered from DNA oxidative attacks. Real-time PCR analysis of enzymatic scavengers expressed in the mouse epididymis indicated that the cauda epididymidis epithelium of Gpx5-null male mice mounted an antioxidant response to cope with an excess of ROS. These observations suggest that GPX5 is a potent antioxidant scavenger in the luminal compartment of the mouse cauda epididymidis that protects spermatozoa from oxidative injuries that could compromise their integrity and, consequently, embryo viability.