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Inhibitory G alpha (GNAI or Gαi) proteins are critical for the polarized morphogenesis of sensory hair cells and for hearing. The extent and nature of their actual contributions remains unclear, however, as previous studies did not investigate all GNAI proteins and included non-physiological approaches. Pertussis toxin can downregulate functionally redundant GNAI1, GNAI2, GNAI3 and GNAO proteins, but may also induce unrelated defects. Here we directly and systematically determined the role(s) of each individual GNAI protein in mouse auditory hair cells. GNAI2 and GNAI3 are similarly polarized at the hair cell apex with their binding partner GPSM2, whereas GNAI1 and GNAO are neither detected nor polarized. InGnai3mutants, GNAI2 progressively fails to fully occupy the subcellular compartments where GNAI3 is missing. In contrast, GNAI3 can fully compensate for the loss of GNAI2 and is essential for hair bundle morphogenesis and auditory function. Simultaneous inactivation ofGnai2andGnai3recapitulates for the first time two distinct types of defects only observed so far with pertussis toxin: 1) a delay or failure of the basal body to migrate off-center in prospective hair cells, and 2) a reversal in the orientation of some hair cell types. We conclude that GNAI proteins are critical for hair cells to break planar symmetry and to orient properly before GNAI2/3 regulate hair bundle morphogenesis with GPSM2.

KM curve of 100 cell injection, quantization of Ki67 and Casp3 staining, and self-renewal of TICs.

The recent identification of cancer stem cells (CSCs) in multiple human cancers provides a new inroad to understanding tumorigenesis at the cellular level. CSCs are defined by their characteristics of self-renewal, multipotentiality, and tumor initiation upon transplantation. By testing for these defining characteristics, we provide evidence for the existence of CSCs in a transgenic mouse model of glioma, S100β-verbB;Trp53. In this glioma model, CSCs are enriched in the side population (SP) cells. These SP cells have enhanced tumor-initiating capacity, self-renewal, and multipotentiality compared with non-SP cells from the same tumors. Furthermore, gene expression analysis comparing fluorescence-activated cell sorting–sorted cancer SP cells to non-SP cancer cells and normal neural SP cells identified 45 candidate genes that are differentially expressed in glioma stem cells. We validated the expression of two genes from this list (S100a4 and S100a6) in primary mouse gliomas and human glioma samples. Analyses of xenografted human glioblastoma multiforme cell lines and primary human glioma tissues show that S100A4 and S100A6 are expressed in a small subset of cancer cells and that their abundance is positively correlated to tumor grade. In conclusion, this study shows that CSCs exist in a mouse glioma model, suggesting that this model can be used to study the molecular and cellular characteristics of CSCs in vivo and to further test the CSC hypothesis. [Cancer Res 2008;68(24):10051–9]

Supplementary Figure 3 from Cancer Stem Cells Are Enriched in the Side Population Cells in a Mouse Model of Glioma

Supplementary Figure Legends from Epigenetic States of Cells of Origin and Tumor Evolution Drive Tumor-Initiating Cell Phenotype and Tumor Heterogeneity

KM survival curve, quantitation of differentiated cells, and H&E of tumorsphere induced tumors.

IPA analysis.

Supplementary Figure 2 from Cancer Stem Cells Are Enriched in the Side Population Cells in a Mouse Model of Glioma

Supplementary Figure 1 from Cancer Stem Cells Are Enriched in the Side Population Cells in a Mouse Model of Glioma

Genes that are differentially expressed among the subtypes.

FISH results from Ptch tumors that progressed to the GFI subtype during in vivo passaging.

Supplementary Tables 1-5 from Cancer Stem Cells Are Enriched in the Side Population Cells in a Mouse Model of Glioma

Supplementary Methods

H&E of tumor derived from injecting SMO;GFAP_cre tumor spheres.

A central confounding factor in the development of targeted therapies is tumor cell heterogeneity, particularly in tumor-initiating cells (TIC), within clinically identical tumors. Here, we show how activation of the Sonic Hedgehog (SHH) pathway in neural stem and progenitor cells creates a foundation for tumor cell evolution to heterogeneous states that are histologically indistinguishable but molecularly distinct. In spontaneous medulloblastomas that arise in Patched (Ptch)+/− mice, we identified three distinct tumor subtypes. Through cell type–specific activation of the SHH pathway in vivo, we determined that different cells of origin evolved in unique ways to generate these subtypes. Moreover, TICs in each subtype had distinct molecular and cellular phenotypes. At the bulk tumor level, the three tumor subtypes could be distinguished by a 465-gene signature and by differential activation levels of the ERK and AKT pathways. Notably, TICs from different subtypes were differentially sensitive to SHH or AKT pathway inhibitors, highlighting new mechanisms of resistance to targeted therapies. In summary, our results show how evolutionary processes act on distinct cells of origin to contribute to tumoral heterogeneity, at both bulk tumor and TIC levels. Cancer Res; 74(17); 4864–74. ©2014 AACR.

Expression patterns of the SHH pathway genes in spontaneous Ptch tumors.

Rhomboid proteases, first discovered in Drosophila, are intramembrane serine proteases. Members of the rhomboid protein family that are catalytically deficient are known as inactive rhomboids (iRhoms). iRhoms have been implicated in wound healing, cancer, and neurological disorders such as Alzheimer’s and Parkinson’s diseases, inflammation, and skin diseases. The past decade of mouse research has shed new light on two key protein domains of iRhoms—the cytosolic N-terminal domain and the transmembrane dormant peptidase domain—suggesting new ways to target multiple intracellular signaling pathways. This review focuses on recent advances in uncovering the unique functions of iRhom protein domains in normal growth and development, growth factor signaling, and inflammation, with a perspective on future therapeutic opportunities.

The CD27–CD70 costimulatory pathway is essential for the full activation of T cells, but some studies show that blocking this pathway exacerbates certain autoimmune disorders. In this study, we report on the impact of CD27–CD70 signaling on disease progression in the NOD mouse model of type 1 diabetes (T1D). Specifically, our data demonstrate that CD70 ablation alters thymocyte selection and increases circulating T cell levels. CD27 signaling was particularly important for the thymic development and peripheral homeostasis of Foxp3+Helios+ regulatory T cells, which likely accounts for our finding that CD70-deficient NOD mice develop more-aggressive T1D onset. Interestingly, we found that CD27 signaling suppresses the thymic development and effector functions of T1D-protective invariant NKT cells. Thus, rather than providing costimulatory signals, the CD27–CD70 axis may represent a coinhibitory pathway for this immunoregulatory T cell population. Moreover, we showed that a CD27 agonist Ab reversed the effects of CD70 ablation, indicating that the phenotypes observed in CD70-deficient mice were likely due to a lack of CD27 signaling. Collectively, our results demonstrate that the CD27–CD70 costimulatory pathway regulates the differentiation program of multiple T cell subsets involved in T1D development and may be subject to therapeutic targeting.

The development of mouse models of human disease and synthetic biology research by targeted transgenesis of large DNA constructs represent a significant genetic engineering hurdle. We developed an efficient, precise, single-copy integration of large transgenes directly into zygotes using multiple mouse genetic backgrounds. We used in vivo Bxb1 mediated recombinase-mediated cassette exchange (RMCE) with a transgene "landing pad" composed of dual heterologous Bxb1 attachment (att) sites in cis, within the Gt(ROSA)26Sor safe harbor locus. RMCE of donor was achieved by microinjection of vector DNA carrying cognate attachment sites flanking the donor transgene with Bxb1-integrase mRNA. This approach achieves perfect vector-free integration of donor constructs at efficiencies 40% with up to ~ 43 kb transgenes. Coupled with a nanopore-based Cas9-targeted sequencing (nCATS), complete verification of precise insertion sequence was achieved. As a proof-of-concept we describe the development of C57BL/6J and NSG Krt18-ACE2 models for SARS-CoV2 research with verified heterozygous N1 animals within ~ 4 months. Additionally, we created a series of mice with diverse backgrounds carrying a single att site including FVB/NJ, PWK/PhJ, NOD/ShiLtJ, CAST/EiJ and DBA/2J allowing for rapid transgene insertion. Combined, this system enables predictable, rapid development with simplified characterization of precisely targeted transgenic animals across multiple genetic backgrounds.

Known as the guardian of the genome, transformation-related protein 53 (TRP53) is a well -known tumor suppressor. Here, we describe a novel TRP53 deficient mouse model on a tumor prone background—SJL/J mice. The absence of TRP53 (TRP53 nullizygosity) leads to a shift in the tumor spectrum from a non-Hodgkin’s-like disease to thymic lymphomas and testicular teratomas at a very rapid tumor onset averaging ~12 weeks of age. In haplotype studies, comparing tumor prone versus tumor resistant Trp53 null mouse strains, we found that other tumor suppressor, DNA repair and/or immune system genes modulate tumor incidence in TRP53 null strains, suggesting that even a strong tumor suppressor such as TRP53 is modulated by genetic background. Due to their rapid development of tumors, the SJL/J TRP53 null mice generated here can be used as an efficient chemotherapy or immunotherapy screening mouse model.

Item does not contain fulltext BACKGROUND: Lithium, mainstay treatment for bipolar disorder, causes nephrogenic diabetes insipidus and hypercalcemia in about 20% and 10% of patients, respectively, and may lead to acidosis. These adverse effects develop in only a subset of patients treated with lithium, suggesting genetic factors play a role. METHODS: To identify susceptibility genes for lithium-induced adverse effects, we performed a genome-wide association study in mice, which develop such effects faster than humans. On day 8 and 10 after assigning female mice from 29 different inbred strains to normal chow or lithium diet (40 mmol/kg), we housed the animals for 48 hours in metabolic cages for urine collection. We also collected blood samples. RESULTS: In 17 strains, lithium treatment significantly elevated urine production, whereas the other 12 strains were not affected. Increased urine production strongly correlated with lower urine osmolality and elevated water intake. Lithium caused acidosis only in one mouse strain, whereas hypercalcemia was found in four strains. Lithium effects on blood pH or ionized calcium did not correlate with effects on urine production. Using genome-wide association analyses, we identified eight gene-containing loci, including a locus containing Acer2, which encodes a ceramidase and is specifically expressed in the collecting duct. Knockout of Acer2 led to increased susceptibility for lithium-induced diabetes insipidus development. CONCLUSIONS: We demonstrate that genome-wide association studies in mice can be used successfully to identify susceptibility genes for development of lithium-induced adverse effects. We identified Acer2 as a first susceptibility gene for lithium-induced diabetes insipidus in mice. 01 december 2019

NOD-SCID IL2rγcnull (NSG) mice are widely used to model human immune cell development because they are more permissive for human hematopoietic cell engraftment and reconstitution than NOD-SCID mice. While increased human reconstitution in the blood of NSG mice has been attributed to the absence of mouse NK cells, deletion of the common gamma chain (γc) limits development of lymphoid tissue inducer cells and precludes development of normal secondary lymphoid structures. The disorganized lymphoid tissue leads to compromised human T cell-B cell interactions and results in variable human immune cell function in human immune system (HIS) NSG compared to NOD-SCID mice. We attempted to remove mouse NK cells from NOD-SCID mice while retaining other γc-dependent cytokine responses by targeted disruption of the mouse genomic IL15RA locus with CRISPR/Cas9. IL15Rα is required for the development, function and survival of NK cells. NOD-SCID IL15Rα-/- mice showed reductions in NK cells and NK cell function. However, NOD-SCID IL15Rα-/- mice demonstrated accelerated thymic lymphomagenesis and showed earlier mortality compared to NOD-SCID mice. This result suggests that mouse NK cells are important to delay lymphoma development in NOD-SCID mice. We transplanted thymectomized NOD-SCID ILl5Rα-/- mice with human fetal liver CD34+ cells and thymus to determine if these mice supported engraftment and development of a transplanted HIS. Surprisingly, we found that peripheral human engraftment was inferior (mean 0.05% of lymphocytes) to that in both NOD-SCID (mean, 10.5% of lymphocytes) and NSG (mean, 54% of lymphocytes) mice. These results indicate that NOD-SCID IL15Rα-/- mice are not permissive for human CD34+ cell engraftment.

Sphingolipids have been implicated in mammalian placental development and function, but their regulation in the placenta remains unclear. Herein we report that alkaline ceramidase 2 (ACER2) plays a key role in sustaining the integrity of the placental vasculature by regulating the homeostasis of sphingolipids in mice. The mouse alkaline ceramidase 2 gene (Acer2) is highly expressed in the placenta between embryonic day (E) 9.5 and E12.5. Acer2 deficiency in both the mother and fetus decreases the placental levels of sphingolipids, including sphingoid bases (sphingosine and dihydrosphingosine) and sphingoid base-1-phosphates ( sphingosine-1-phophate and dihydrosphingosine-1-phosphate) and results in the in utero death of ≈ 50% of embryos at E12.5 whereas Acer2 deficiency in either the mother or fetus has no such effects. Acer2 deficiency causes hemorrhages from the maternal vasculature in the junctional and/or labyrinthine zones in E12.5 placentas. Moreover, hemorrhagic but not non-hemorrhagic Acer2-deficient placentas exhibit an expansion of parietal trophoblast giant cells with a concomitant decrease in the area of the fetal blood vessel network in the labyrinthine zone, suggesting that Acer2 deficiency results in embryonic lethality due to the atrophy of the fetal blood vessel network in the placenta. Taken together, these results suggest that ACER2 sustains the integrity of the placental vasculature by controlling the homeostasis of sphingolipids in mice.

Immunodeficient mice engrafted with human peripheral blood mononuclear cells (PBMCs) support preclinical studies of human pathogens, allograft rejection, and human T-cell function. However, a major limitation of PBMC engraftment is development of acute xenogeneic graft-versus-host disease (GVHD) due to human T-cell recognition of murine major histocompatibility complex (MHC). To address this, we created 2 NOD-scid IL-2 receptor subunit γ (IL2rg)(null) (NSG) strains that lack murine MHC class I and II [NSG–β-2-microglobulin (B2M)(null) (IA IE)(null) and NSG-(K(b) D(b))(null) (IA(null))]. We observed rapid human IgG clearance in NSG-B2M(null) (IA IE)(null) mice whereas clearance in NSG-(K(b) D(b))(null) (IA(null)) mice and NSG mice was comparable. Injection of human PBMCs into both strains enabled long-term engraftment of human CD4(+) and CD8(+) T cells without acute GVHD. Engrafted human T-cell function was documented by rejection of human islet allografts. Administration of human IL-2 to NSG-(K(b) D(b))(null) (IA(null)) mice via adeno-associated virus vector increased human CD45(+) cell engraftment, including an increase in human regulatory T cells. However, high IL-2 levels also induced the development of GVHD. These data document that NSG mice deficient in murine MHC support studies of human immunity in the absence of acute GVHD and enable evaluation of human antibody therapeutics targeting human T cells.—Brehm, M. A., Kenney, L. L., Wiles, M. V., Low, B. E., Tisch, R. M., Burzenski, L., Mueller, C., Greiner, D. L., Shultz, L. D. Lack of acute xenogeneic graft-versus-host disease, but retention of T-cell function following engraftment of human peripheral blood mononuclear cells in NSG mice deficient in MHC class I and II expression.

Distal enhancers are thought to play important roles in the spatiotemporal regulation of gene expression during embryonic development, but few predicted enhancer elements have been shown to affect transcription of their endogenous genes or to alter phenotypes when disrupted. Here, we demonstrate that a 123.6-kb deletion within the mouse Slc25a13 gene is associated with reduced transcription of Dlx5, a gene located 660 kb away. Mice homozygous for the Slc25a13 deletion mutation [named hyperspin (hspn)] have malformed inner ears and are deaf with balance defects, whereas previously reported Slc25a13 knockout mice showed no phenotypic abnormalities. Inner ears of Slc25a13hspn/hspn mice have malformations similar to those of Dlx5−/− embryos, and Dlx5 expression is severely reduced in the otocyst but not the branchial arches of Slc25a13hspn/hspn embryos, indicating that the Slc25a13hspn deletion affects otic-specific enhancers of Dlx5. In addition, transheterozygous Slc25a13+/hspn Dlx5+/− mice exhibit noncomplementation with inner ear dysmorphologies similar to those of Slc25a13hspn/hspn and Dlx5−/−embryos, verifying a cis-acting effect of the Slc25a13hspn deletion on Dlx5 expression. CRISPR/Cas9-mediated deletions of putative enhancer elements located within the Slc25a13hspn deleted region failed to phenocopy the defects of Slc25a13hspn/hspn mice, suggesting the possibility of multiple enhancers with redundant functions. Our findings in mice suggest that analogous enhancer elements in the human SLC25A13 gene may regulate DLX5 expression and underlie the hearing loss that is associated with split-hand/-foot malformation 1 syndrome. Slc25a13hspn/hspn mice provide a new animal model for studying long-range enhancer effects on Dlx5 expression in the developing inner ear.

Sphingosine-1-phosphate (S1P) plays important roles in cardiovascular development and immunity. S1P is abundant in plasma because erythrocytes-the major source of S1P-lack any S1P-degrading activity; however, much remains unclear about the source of the plasma S1P precursor, sphingosine (SPH), derived mainly from the hydrolysis of ceramides by the action of ceramidases that are encoded by 5 distinct genes, acid ceramidase 1 ( ASAH1)/ Asah1, ASAH2/ Asah2, alkaline ceramidase 1 ( ACER1)/ Acer1, ACER2/ Acer2, and ACER3/ Acer3, in humans/mice. Previous studies have reported that knocking out Asah1 or Asah2 failed to reduce plasma SPH and S1P levels in mice. In this study, we show that knocking out Acer1 or Acer3 also failed to reduce the blood levels of SPH or S1P in mice. In contrast, knocking out Acer2 from either whole-body or the hematopoietic lineage markedly decreased the blood levels of SPH and S1P in mice. Of interest, knocking out Acer2 from whole-body or the hematopoietic lineage also markedly decreased the levels of dihydrosphingosine (dhSPH) and dihydrosphingosine-1-phosphate (dhS1P) in blood. Taken together, these results suggest that ACER2 plays a key role in the maintenance of high plasma levels of sphingoid base-1-phosphates-S1P and dhS1P-by controlling the generation of sphingoid bases-SPH and dhSPH-in hematopoietic cells.-Li, F., Xu, R., Low, B. E., Lin, C.-L., Garcia-Barros, M., Schrandt, J., Mileva, I., Snider, A., Luo, C. K., Jiang, X.-C., Li, M.-S., Hannun, Y. A., Obeid, L. M., Wiles, M. V., Mao, C. Alkaline ceramidase 2 is essential for the homeostasis of plasma sphingoid bases and their phosphates.

Purpose To determine the molecular basis of lesion development in a murine model of spontaneous retinal vascularization, rnv3 (retinal vascularization 3, aka JR5558). Methods Disease progression of rnv3 was examined in longitudinal studies by clinical evaluation, electroretinography (ERG) and light microscopy analyses. The chromosomal position for the recessive rnv3 mutation was determined by DNA pooling and genome-wide linkage analysis. The causative mutation was discovered by comparison of whole exome sequences of rnv3 mutant and wild-type (WT) controls. In order to confirm the causative mutation, transcription activator-like effector nuclease (TALEN)-mediated oligonucleotide directed repair (ODR) was utilized to correct the mutant allele. Phenotypic correction was assessed by fundus imaging and optical coherence tomography of live mice. Results rnv3 exhibits early-onset, multifocal depigmented retinal lesions observable by fundus examination starting at 18 days of age. The retinal lesions are associated with fluorescein leakage around 25 days of age, with peak leakage at about 4 weeks of age. ERG responses deteriorate as rnv3 mutants age, concomitant with progressive photoreceptor disruption and loss that is observable by histology. Genetic analysis localized rnv3 to mouse chromosome (Chr) 1. By high throughput sequencing of a whole exome capture library of a rnv3/rnv3 mutant and subsequent sequence analysis, a single base deletion (del) in the Crb1 [crumbs family member 1] gene, which was previously reported to cause retinal degeneration 8, was identified. The TALEN-mediated ODR rescued the posterior segment vascularization phenotype; heterozygous Crb1rd8+em1Boc/Crb1rd8 and homozygous Crb1rd8+em1Boc/Crb1rd8+em1Boc mice showed a normal retinal phenotype. Additionally, six novel disruptions of Crb1 that were generated through aberrant non-homologous end joining induced by TALEN exhibited variable levels of vascularization, suggesting allelic effects. Conclusions The rnv3 model and the models of six novel disruptions of Crb1 are all reliable, novel mouse models for the study of both early and late events associated with posterior segment vascularization and can also be used to test the effects of pharmacological targets for treating human ocular vascular disorders. Further study of these models may provide a greater understanding about how different Crb1 alleles result in aberrant angiogenesis.

Mice are the most commonly used model organisms to study human disease. Many genetic human diseases can be recapitulated by modifying the mouse genome allowing the testing of existing and novel therapeutics, including combinatorial therapeutics, without putting humans at risk. Specifically, the development of "humanized" mice, that is, severely immunodeficient mice engrafted with functional human hematopoietic and immune cells and tissues, has revolutionized our ability to study and model human diseases in preclinical in vivo systems. Until recently it has been challenging to develop strains of humanized mice with targeted mutations or that transgenically express human genes with site-specific mutations, and can permit optimal growth of functional human cells and tissues. However, recent advances in targeted nuclease-based genetic engineering have enabled precise modification and development of humanized mouse models at an unprecedented pace. These modifications permit optimal growth of functional human cells and tissues and can be used to replicate human genetically determined diseases. J. Cell. Biochem. 118: 3043-3048, 2017. © 2017 Wiley Periodicals, Inc.

Mice have been excellent surrogates for studying neutrophil biology and, furthermore, murine models of human disease have provided fundamental insights into the roles of human neutrophils in innate immunity. The emergence of novel humanized mice and high-diversity mouse populations offers the research community innovative and powerful platforms for better understanding, respectively, the mechanisms by which human neutrophils drive pathogenicity, and how genetic differences underpin the variation in neutrophil biology observed among humans. Here, we review key examples of these new resources. Additionally, we provide an overview of advanced genetic engineering tools available to further improve such murine model systems, of sophisticated neutrophil-profiling technologies, and of multifunctional nanoparticle (NP)-based neutrophil-targeting strategies.

Pseudoxanthoma elasticum, a prototype of heritable multisystem ectopic mineralization disorders, is caused by mutations in the ABCC6 gene encoding a putative efflux transporter, ABCC6. The phenotypic spectrum of pseudoxanthoma elasticum varies, and the correlation between genotype and phenotype has not been established. To identify genetic modifiers, we performed quantitative trait locus analysis in inbred mouse strains that carry the same hypomorphic allele in Abcc6 yet with highly variable ectopic mineralization phenotypes of pseudoxanthoma elasticum. Abcc6 was confirmed as a major determinant for ectopic mineralization in multiple tissues. Integrative analysis using functional genomics tools that included GeneWeaver, String, and Mouse Genome Informatics identified a total of nine additional candidate modifier genes that could influence the organ-specific ectopic mineralization phenotypes. Integration of the candidate genes into the existing ectopic mineralization gene network expands the current knowledge on the complexity of the network that, as a whole, governs ectopic mineralization in soft connective tissues.

BACKGROUND: Lithium, mainstay treatment for bipolar disorder, causes nephrogenic diabetes insipidus and hypercalcemia in about 20% and 10% of patients, respectively, and may lead to acidosis. These adverse effects develop in only a subset of patients treated with lithium, suggesting genetic factors play a role. METHODS: To identify susceptibility genes for lithium-induced adverse effects, we performed a genome-wide association study in mice, which develop such effects faster than humans. On day 8 and 10 after assigning female mice from 29 different inbred strains to normal chow or lithium diet (40 mmol/kg), we housed the animals for 48 hours in metabolic cages for urine collection. We also collected blood samples. RESULTS: In 17 strains, lithium treatment significantly elevated urine production, whereas the other 12 strains were not affected. Increased urine production strongly correlated with lower urine osmolality and elevated water intake. Lithium caused acidosis only in one mouse strain, whereas hypercalcemia was found in four strains. Lithium effects on blood pH or ionized calcium did not correlate with effects on urine production. Using genome-wide association analyses, we identified eight gene-containing loci, including a locus containing Acer2, which encodes a ceramidase and is specifically expressed in the collecting duct. Knockout of Acer2 led to increased susceptibility for lithium-induced diabetes insipidus development. CONCLUSIONS: We demonstrate that genome-wide association studies in mice can be used successfully to identify susceptibility genes for development of lithium-induced adverse effects. We identified Acer2 as a first susceptibility gene for lithium-induced diabetes insipidus in mice.

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-Fcgrt(tm1Dcr) 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(-/-) hFcRn(Tg/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.

Distal enhancers are thought to play important roles in the spatiotemporal regulation of gene expression during embryonic development, but few predicted enhancer elements have been shown to affect transcription of their endogenous genes or to alter phenotypes when disrupted. Here, we demonstrate that a 123.6-kb deletion within the mouse Slc25a13 gene is associated with reduced transcription of Dlx5, a gene located 660 kb away. Mice homozygous for the Slc25a13 deletion mutation [named hyperspin (hspn)] have malformed inner ears and are deaf with balance defects, whereas previously reported Slc25a13 knockout mice showed no phenotypic abnormalities. Inner ears of Slc25a13(hspn/hspn) mice have malformations similar to those of Dlx5(−)(/)(−) embryos, and Dlx5 expression is severely reduced in the otocyst but not the branchial arches of Slc25a13(hspn/hspn) embryos, indicating that the Slc25a13(hspn) deletion affects otic-specific enhancers of Dlx5. In addition, transheterozygous Slc25a13(+/hspn) Dlx5(+/)(−) mice exhibit noncomplementation with inner ear dysmorphologies similar to those of Slc25a13(hspn/hspn) and Dlx5(−)(/)(−)embryos, verifying a cis-acting effect of the Slc25a13(hspn) deletion on Dlx5 expression. CRISPR/Cas9-mediated deletions of putative enhancer elements located within the Slc25a13(hspn) deleted region failed to phenocopy the defects of Slc25a13(hspn/hspn) mice, suggesting the possibility of multiple enhancers with redundant functions. Our findings in mice suggest that analogous enhancer elements in the human SLC25A13 gene may regulate DLX5 expression and underlie the hearing loss that is associated with split-hand/-foot malformation 1 syndrome. Slc25a13(hspn/hspn) mice provide a new animal model for studying long-range enhancer effects on Dlx5 expression in the developing inner ear.

There are ~350 million chronic carriers of hepatitis B (HBV). While a prophylactic vaccine and drug regimens to suppress viremia are available, chronic HBV infection is rarely cured. HBV's limited host tropism leads to a scarcity of susceptible small animal models and is a hurdle to developing curative therapies. Mice that support engraftment with human hepatoctyes have traditionally been generated through crosses of murine liver injury models to immunodeficient backgrounds. Here, we describe the disruption of fumarylacetoacetate hydrolase directly in the NOD Rag1-/- IL2RγNULL (NRG) background using zinc finger nucleases. The resultant human liver chimeric mice sustain persistent HBV viremia for >90 days. When treated with standard of care therapy, HBV DNA levels decrease below detection but rebound when drug suppression is released, mimicking treatment response observed in patients. Our study highlights the utility of directed gene targeting approaches in zygotes to create new humanized mouse models for human diseases.

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.

Albumin is a large, highly abundant protein circulating in the blood stream which is regulated and actively recycled via the neonatal Fc receptor (FcRn). In humans this results in serum albumin having an exceptional long half-life of ~21 days. Some time ago it was realized that these intrinsic properties could be harnessed and albumin could be used as a privileged drug delivery vehicle. However, active development of albumin based therapeutics has been hampered by the lack of economic, relevant experimental models which can accurately recapitulate human albumin metabolism and pharmacokinetics. In mice for example, introduced human albumin is not recycled and is catabolized rapidly. This is mainly due to the failure of mouse FcRn to bind human albumin consequently, human albumin has a half-life of only 2-3 days in mice. To overcome this we developed and characterized a humanized mouse model which is null for mouse FcRn and mouse albumin, but is transgenic for, and expressing functional human FcRn. Published data clearly demonstrate that upon injection of human albumin into this model animal that it accurately recapitulates human albumin FcRn dependent serum recycling, with human albumin now having a half-life ~24 days, closely mimicking that observed in humans. In this practical review we briefly review this model and outline its use for pharmacokinetic studies of human albumin.

Highly definable genetically, the humble mouse is the "reagent" mammal of choice with which to probe and begin to understand the human condition in all its complexities. With the recent advance in direct genome editing via targeted nucleases, e.g., TALEN and CRISPR/Cas9, the possibilities in using these sophisticated tools have increased substantially leading to a massive increase in the variety of strain numbers of genetically modified lines. With this increase comes a greater need to economically and creatively manage their numbers. Further, once characterized, lines may be of limited use but still need to be archived in a format allowing their rapid resurrection. Further, maintaining colonies on "the shelf" is financially draining and carries potential risks including natural disaster loss, disease, and strain contamination. Here we outline a simple and economic protocol to cryopreserve mouse sperm from many different genetic backgrounds, and outline its recovery via in vitro fertilization (IVF). The combined use of sperm cryopreservation and IVF now allows a freedom and versatility in mouse management facilitating rapid line close down with the option to later recover and rapidly expand as needed.

Mouse models continue to be powerful tools in preclinical oncology and immuno-oncology studies. The development of the inbred highly immunocompromised mouse strain NSG (NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ) has dramatically improved the ability to work with human tumors in an organismal context using xenograft approaches. Furthermore, technical innovations have expanded the utility of this personalized tumor platform via the reconstitution of parts of the human immune system. An example of this is the NSG-SGM3 inbred mouse strain, in which transgenes expressing human IL3, GMCSF and SCF have been stably integrated into the NSG background. This NSG “derivative” strain can effectively support the stable engraftment of human myeloid lineages and the regulatory T cell populations. Direct genetic modification of the NSG genome in mouse embryos was a key technical capability to attain this NSG “derivative” strain rapidly and avoid extensive breeding. Modification of NSG and derivative strains represent an opportunity to optimize the engraftment of other human lineages. The ability to apply modern genome modification technology to different inbred mouse strains is highly desirable but must overcome two major technical hurdles - 1) Reproductive biology constraints that prevent live born progeny after reagent delivery methods such as microinjection or electroporation and 2) Cell intrinsic DNA-repair processes that are a prerequisite enabling gene editing tools. Given the SCID mutation (Prkdcscid) in the NSG background compromises some DNA repair pathways, it was not clear whether nuclease assisted recombination technologies would be a robust tool to modify NSG (and derivatives) genomes at the zygote stage. Here, we demonstrate that CRISPR-Cas9 is an effective tool for introducing genome modifications in NSG and present a specific example using the Hprt locus. Furthermore, we will present general experience with genomic modifications using NSG, and its derivatives, to emphasize direct modification of these genomes is technically feasible and is enabling the rapid sequential genetic tailoring in this high value cancer model strain. Citation Format: David S. Grass, F William Buaas, Michael V. Wiles, Benjamin E. Low, Peter Kutny. Gene editing in the NSG mouse strain and its genetic derivatives [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5111.

The NSG (NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ) mouse strain is highly immunodeficient and has dramatically improved the ability to work with human tumors in an organismal context using xenograft approaches. Technical innovations have expanded the utility of this personalized tumor platform via the reconstitution of parts of the human immune system. An example of this is the NSG-SGM3 inbred mouse strain, in which transgenes expressing human IL3, GMCSF and SCF have been stably integrated into the NSG background, and can effectively support the stable engraftment of human myeloid lineages and the regulatory T cell populations. Direct modification of the NSG genome in mouse embryos was a key to attain this NSG “derivative” strain rapidly and avoid extensive breeding. Modification of NSG, which is built on the parental NOD/ShiLtJ background, a commonly utilized polygenic model of autoimmune type 1 diabetes, can optimize the engraftment of other human lineages. Modern genome modification technology must overcome strain specific characteristics. DNA repair is involved in creating mutations by resolving double strand breaks. The SCID mutation (Prkdcscid) in the NSG background compromises some DNA repair pathways. It was not clear whether nuclease assisted recombination technologies would be a robust tool to modify NSG genomes at the zygote stage. We demonstrate CRISPR-Cas9 is an effective tool for introducing genome modifications in NSG and present a specific example using the Hprt locus. We will also present our experience modifying NSG and its derivatives, demonstrating that direct modification of these genomes is technically feasible and is enabling the rapid sequential genetic tailoring in this high value immunodeficient strain.