Generation Of Disease Models Research Articles

Modeling diseases in multiple mouse strains for precision medicine studies.

The genetic basis of the phenotypic variability observed in patients can be studied in mice by generating disease models through genetic or chemical interventions in many genetic backgrounds where the clinical phenotypes can be assessed and used for genome-wide association studies (GWAS). This is particularly relevant for rare disorders, where patients sharing identical mutations can present with a wide variety of symptoms, but there are not enough number of patients to ensure statistical power of GWAS. Inbred strains are homozygous for each loci, and their single nucleotide polymorphisms catalogs are known and freely available, facilitating the bioinformatics and reducing the costs of the study, since it is not required to genotype every mouse. This kind of approach can be applied to pharmacogenomics studies as well.

Genome editing: the road of CRISPR/Cas9 from bench to clinic.

Molecular scissors engineered for site-specific modification of the genome hold great promise for effective functional analyses of genes, genomes and epigenomes and could improve our understanding of the molecular underpinnings of disease states and facilitate novel therapeutic applications. Several platforms for molecular scissors that enable targeted genome engineering have been developed, including zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and, most recently, clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated-9 (Cas9). The CRISPR/Cas9 system's simplicity, facile engineering and amenability to multiplexing make it the system of choice for many applications. CRISPR/Cas9 has been used to generate disease models to study genetic diseases. Improvements are urgently needed for various aspects of the CRISPR/Cas9 system, including the system's precision, delivery and control over the outcome of the repair process. Here, we discuss the current status of genome engineering and its implications for the future of biological research and gene therapy.

The contribution of mouse models to the understanding of constitutional thrombocytopenia.

Constitutional thrombocytopenias result from platelet production abnormalities of hereditary origin. Long misdiagnosed and poorly studied, knowledge about these rare diseases has increased considerably over the last twenty years due to improved technology for the identification of mutations, as well as an improvement in obtaining megakaryocyte culture from patient hematopoietic stem cells. Simultaneously, the manipulation of mouse genes (transgenesis, total or conditional inactivation, introduction of point mutations, random chemical mutagenesis) have helped to generate disease models that have contributed greatly to deciphering patient clinical and laboratory features. Most of the thrombocytopenias for which the mutated genes have been identified now have a murine model counterpart. This review focuses on the contribution that these mouse models have brought to the understanding of hereditary thrombocytopenias with respect to what was known in humans. Animal models have either i) provided novel information on the molecular and cellular pathways that were missing from the patient studies; ii) improved our understanding of the mechanisms of thrombocytopoiesis; iii) been instrumental in structure-function studies of the mutated gene products; and iv) been an invaluable tool as preclinical models to test new drugs or develop gene therapies. At present, the genetic determinants of thrombocytopenia remain unknown in almost half of all cases. Currently available high-speed sequencing techniques will identify new candidate genes, which will in turn allow the generation of murine models to confirm and further study the abnormal phenotype. In a complementary manner, programs of random mutagenesis in mice should also identify new candidate genes involved in thrombocytopenia.

Abstract 670: The Jackson Laboratory Repository: Resource for mouse models of human cancer

Abstract The mouse is a versatile model organism whose value has been augmented by recent biotechnological innovations in genome editing. While standard inbred and spontaneous mouse strains are still widely utilized, developments in genetic engineering technologies have significantly broadened the strategies available to researchers to generate specific disease models with preclinical applications. The Jackson Laboratory (JAX) has been serving the scientific community as a resource for mouse strains relevant to human disease for more than seven decades. The JAX Repository consists of over 8,000 mouse strains, many with applications for modeling human cancer. A wide selection of models are now available that have applications as hosts for cancer xenograft modeling. Strains have become increasingly specialized for specific purposes: optimized support of human and murine hematopoietic cell engraftment, reduced xenogeneic graft-versus-host disease response, engraftment of human hematopoietic stem cells without irradiation, and immunological tolerance to firefly luciferase and enhanced green fluorescent protein. Mouse strains that recapitulate aspects of specific diseases, such as Chordoma, are also available. In response to the promising potential of CRISPR/cas9 technology, a variety of cas9-expressing lines (constitutive and Cre-inducible expression) are included in the JAX collection for more convenient generation of disease models. Whereas a gene editing approach using viral delivery of cas9 is burdened by packaging size limits, these cas9-expressing mice only require a specific single guide RNA for generating mutations. cas9 expressing mice are available on several congenic strain backgrounds and involve constructs knocked into two different loci, Gt(ROSA)26Sor or Igs2 (also known as Hipp11). The location of the Igs2 locus on chromosome 11 (cytoband A1 at ∼3cM between the Eif4enif1 and Drg1 loci) occurs within an intergenic region which reduces possible disruption of other endogenous genes. All Repository mouse strains are cryopreserved and monitored by a robust quality control program to confirm mutation identity and genetic background. A new JAXMice website search interface facilitates queries by disease term as well as by keyword and strain, allele, and gene name or synonym (www.jax.org). Donating a strain to the Repository fulfills the requirements of the NIH's policy for the sharing of research reagents. Researchers wishing to have strains considered for inclusion in the Repository can find the submission form available at: www.jax.org/donate-a-mouse. The Jackson Laboratory Repository is supported by the NIH, The Howard Hughes Medical Institute, and other private charitable foundations. Citation Format: Deborah Boswell, Steve Rockwood, Mike Sasner, The JAX Repository Team. The Jackson Laboratory Repository: Resource for mouse models of human cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 670.

Generation of obese rat model by transcription activator-like effector nucleases targeting the leptin receptor gene

The laboratory rat is a valuable mammalian model organism for basic research and drug discovery. Here we demonstrate an efficient methodology by applying transcription activator-like effector nucleases (TALENs) technology to generate Leptin receptor (Lepr) knockout rats on the Sprague Dawley (SD) genetic background. Through direct injection of in vitro transcribed mRNA of TALEN pairs into SD rat zygotes, somatic mutations were induced in two of three resulting pups. One of the founders carrying bi-allelic mutation exhibited early onset of obesity and infertility. The other founder carried a chimeric mutation which was efficiently transmitted to the progenies. Through phenotyping of the resulting three lines of rats bearing distinct mutations in the Lepr locus, we found that the strains with a frame-shifted or premature stop codon mutation led to obesity and metabolic disorders. However, no obvious defect was observed in a strain with an in-frame 57 bp deletion in the extracellular domain of Lepr. This suggests the deleted amino acids do not significantly affect Lepr structure and function. This is the first report of generating the Lepr mutant obese rat model in SD strain through a reverse genetic approach. This suggests that TALEN is an efficient and powerful gene editing technology for the generation of disease models.

A method for using real world data in breast cancer modeling

ObjectivesToday, hospitals and other health care-related institutions are accumulating a growing bulk of real world clinical data. Such data offer new possibilities for the generation of disease models for the health economic evaluation. In this article, we propose a new approach to leverage cancer registry data for the development of Markov models. Records of breast cancer patients from a clinical cancer registry were used to construct a real world data driven disease model. MethodsWe describe a model generation process which maps database structures to disease state definitions based on medical expert knowledge. Software was programmed in Java to automatically derive a model structure and transition probabilities. We illustrate our method with the reconstruction of a published breast cancer reference model derived primarily from clinical study data. In doing so, we exported longitudinal patient data from a clinical cancer registry covering eight years. The patient cohort (n=892) comprised HER2-positive and HER2-negative women treated with or without Trastuzumab. ResultsThe models generated with this method for the respective patient cohorts were comparable to the reference model in their structure and treatment effects. However, our computed disease models reflect a more detailed picture of the transition probabilities, especially for disease free survival and recurrence. ConclusionsOur work presents an approach to extract Markov models semi-automatically using real world data from a clinical cancer registry. Health care decision makers may benefit from more realistic disease models to improve health care-related planning and actions based on their own data.

The Development of TALE Nucleases for Biotechnology.

The development of a facile genome engineering technology based on transcription activator-like effector nucleases (TALENs) has led to significant advances in diverse areas of science and medicine. In this review, we provide a broad overview of the development of TALENs and the use of this technology in basic science, biotechnology, and biomedical applications. This includes the discovery of DNA recognition by TALEs, engineering new TALE proteins to diverse targets, general advances in nuclease-based editing strategies, and challenges that are specific to various applications of the TALEN technology. We review examples of applying TALENs for studying gene function and regulation, generating disease models, and developing gene therapies. The current status of genome editing and future directions for other uses of these technologies are also discussed.

Mutations in the VEGFR3 Signaling Pathway Explain 36% of Familial Lymphedema

Lymphedema is caused by dysfunction of lymphatic vessels, leading to disabling swelling that occurs mostly on the extremities. Lymphedema can be either primary (congenital) or secondary (acquired). Familial primary lymphedema commonly segregates in an autosomal dominant or recessive manner. It can also occur in combination with other clinical features. Nine mutated genes have been identified in different isolated or syndromic forms of lymphedema. However, the prevalence of primary lymphedema that can be explained by these genetic alterations is unknown. In this study, we investigated 7 of these putative genes. We screened 78 index patients from families with inherited lymphedema for mutations in FLT4, GJC2, FOXC2, SOX18, GATA2, CCBE1, and PTPN14. Altogether, we discovered 28 mutations explaining 36% of the cases. Additionally, 149 patients with sporadic primary lymphedema were screened for FLT4, FOXC2, SOX18,CCBE1, and PTPN14. Twelve mutations were found that explain 8% of the cases. Still unidentified is the genetic cause of primary lymphedema in 64% of patients with a family history and 92% of sporadic cases. Identification of those genes is important for understanding of etiopathogenesis, stratification of treatments and generation of disease models. Interestingly, most of the proteins that are encoded by the genes mutated in primary lymphedema seem to act in a single functional pathway involving VEGFR3 signaling. This underscores the important role this pathway plays in lymphatic development and function and suggests that the unknown genes also have a role.

Hooked! Modeling human disease in zebrafish

Zebrafish have been widely used as a model system for studying developmental processes, but in the last decade, they have also emerged as a valuable system for modeling human disease. The development and function of zebrafish organs are strikingly similar to those of humans, and the ease of creating mutant or transgenic fish has facilitated the generation of disease models. Here, we highlight the use of zebrafish for defining disease pathways and for discovering new therapies.

Zebrafish embryos and larvae: A new generation of disease models and drug screens

Technological innovation has helped the zebrafish embryo gain ground as a disease model and an assay system for drug screening. Here, we review the use of zebrafish embryos and early larvae in applied biomedical research, using selected cases. We look at the use of zebrafish embryos as disease models, taking fetal alcohol syndrome and tuberculosis as examples. We discuss advances in imaging, in culture techniques (including microfluidics), and in drug delivery (including new techniques for the robotic injection of compounds into the egg). The use of zebrafish embryos in early stages of drug safety-screening is discussed. So too are the new behavioral assays that are being adapted from rodent research for use in zebrafish embryos, and which may become relevant in validating the effects of neuroactive compounds such as anxiolytics and antidepressants. Readouts, such as morphological screening and cardiac function, are examined. There are several drawbacks in the zebrafish model. One is its very rapid development, which means that screening with zebrafish is analogous to "screening on a run-away train." Therefore, we argue that zebrafish embryos need to be precisely staged when used in acute assays, so as to ensure a consistent window of developmental exposure. We believe that zebrafish embryo screens can be used in the pre-regulatory phases of drug development, although more validation studies are needed to overcome industry scepticism. Finally, the zebrafish poses no challenge to the position of rodent models: it is complementary to them, especially in early stages of drug research.

316 INDUCTION OF PIG INDUCED PLURIPOTENT STEM CELLS BY RECOMBINANT PROTEINS ENCODED BY DEFINED FACTORS

Pluripotent cells derived from any differentiated cell type through ectopic expression of transcription factors were designated as induced pluripotent stem (iPS) cells, exhibiting similar morphology and growth properties to embryonic stem (ES) cells besides expressing ES cell marker. Because iPS have the ability to differentiate into all types of cells, iPS cell technology is thought to have enormous potential for generating disease models, drug screening, toxicology, and regenerative medicine. However, for virus-mediated transfection of defined factors, the exogenous genes generally would be randomly inserted into the target cell’s genome, possibly bringing the potential hazard of insertional mutagenesis. Therefore, it is necessary to seek some new methods to induce somatic cell reprogramming without viruses. With instruction from the work of Zhou et al. (2009 Cell Stem Cell 4, 381–384), in the present study we attempted to use defined factors recombinant proteins-carried cell-penetrating peptide for the generation of porcine iPS cells, which would be a benefit for safe applications of iPS cells. Defined factors genes were amplified by PCR with specific primers of 9 arginines (R9) from recombinant plasmid pLL-hOCT4/pSox2/pMyc/pKlf4-EGFP (Yin et al. 2010 Prog. Biochem. Biophys. 37, 607–612) and inserted into prokaryotic expression vector pET-28a-EGFP. After DNA sequencing confirmation, the 4 recombinant plasmids were then transformed into BL21 strains, respectively. After IPTG induction, hOCT4/pSox2/pMyc/pKlf4-R9-EGFP fusion proteins were purified using Novagen His-Bind kit and confirmed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, respectively. Then, defined factors recombinant proteins were added into pig fetal fibroblasts (PFF) medium every 48 h to establish pig iPS cells. The results showed that purified hOCT4/pSox2/pMyc/pKlf4-R9-EGFP fusion proteins could enter into PFF efficiently, and most of them were located in nuclei. The PPF were subcultured in stem cell medium condition and treated with defined factors recombinant proteins for 6 cycles simultaneously; the clear-cut cell colonies were gradually derived. These cells had large translucent nuclei and a high nucleo:cytoplasmic ratio and were positive for AP, Oct4, and Nanog. Detailed characterisation of such induced cells is ongoing. This research would provide new ideas for the induction of porcine somatic cell reprogramming. Y. H. Zhang and H. G. Cao contributed equally. This work was supported by NSFC (30700574 30800784/c120103) and 973 (2009CB941004).

Immunohistochemical detection of transgene expression in the brain using small epitope tags

BackgroundIn vivo overexpression of proteins is a powerful approach to study their biological function, generate disease models or evaluate gene therapy approaches. In order to investigate an exogenously expressed protein, specific and sensitive detection is essential. Unfortunately, antibodies that allow histological detection of the protein of interest are not always readily available. The use of an epitope tag fused to the protein can circumvent this problem as well as provide the possibility to discriminate endogenous from overexpressed proteins. In order to minimize impact on the bioactivity and biodistribution of the overexpressed protein, preference is given to small tags.ResultsIn the present study, we evaluated several small epitope tags together with corresponding anti-tag antibodies for the detection of overexpressed proteins in rat brain, using eGFP as a reference. We generated several lentiviral vectors encoding eGFP with different N-terminally fused small epitope tags (AU1, flag, 3flag, HA, myc and V5). After confirmation of their functionality in cell culture, we injected these lentiviral vectors stereotactically into the striatum of rats and prepared paraformaldehyde fixed floating sections for immunohistochemical analysis. Using multiple antibodies and antibody dilutions per epitope tag, we extensively assessed the efficiency of several anti-tag antibodies for chromogenic immunohistochemical detection of the epitope tagged eGFPs by determining the proportion of immunoreactivity detected by anti-tag antibodies compared to anti-GFP antibody. Using fluorescence immunohistochemistry and confocal microscopy, we also quantified the proportion of eGFP-positive cells detected by anti-tag antibodies. Our results show that all the examined small epitope tags could be detected by anti-tag antibodies both in cell extracts as well as in vivo, although to varying degrees depending on the tag and antibody used. Using the presented protocol, V5/anti-V5 and HA/HA11 tag/antibody combinations provided the most sensitive detection in brain tissue. We confirmed the applicability of these optimized in vivo tag detection conditions for a difficult to detect protein, firefly luciferase (fLuc), using lentiviral vector constructs expressing V5 tagged and 3flag tagged fLuc protein.ConclusionsWe show here that several small epitope tags are useful for immunohistochemical detection of exogenous proteins in vivo. Our study also provides a generic methodology which is broadly applicable for the detection of overexpressed transgenes in mammalian brain tissue.

Ekiden to iPS Cells

When I started a long ekiden—a relay marathon by multiple runners—toward cellular reprogramming about a decade ago, not many teams joined the start of the race. At the time, many laboratories were trying to differentiate embryonic stem (ES) cells into various functional cells, inspired by the report on the isolation of human ES cells1. In contrast, I tried to dedifferentiate somatic cells back to the embryonic state. I was not sure if we could reach our goal in my lifetime, but thanks to diligent colleagues and students in my laboratory (Fig. 1), luck favored us. In August 2006, we announced the generation of ES cell–like stem cells by introducing four transcription factors—Oct3/4, Sox2, Klf4 and c-Myc—into mouse fibroblasts by retroviral vectors2. I named this new type of cells ‘induced pluripotent stem (iPS) cells’. We reported the generation of human iPS cells3 on 20 November 2007, the same day that James Thomson and his colleagues also announced their success in making human iPS cells4. With the ability to differentiate into virtually all types of cells and to grow robustly like ES cells, iPS cells have enormous potential for pharmaceutical and clinical applications5,6. The technology is expected to be used for generating disease models, drug screening, toxicology and regenerative medicine, circumventing two obstacles surrounding ES cells—the ethical controversy over destruction of embryos to isolate ES cells and the risks of immune rejection when ES-derived cells are transplanted into a person. In vitro uses of iPS cells are just around the corner, but there are still many hurdles before their clinical application. However, I believe that strenuous efforts of researchers around the world will make the promises a reality in the not-too-distant future. Today, iPS cell research has become one of the hottest areas in the life sciences, with an increasing number of laboratories worldwide working on the technology. Many new findings have been reported in the short time since the first report was published5,7. I cannot stress enough that without the many studies using mouse and human ES cells that have appeared since the isolation of mouse ES cells in 1981 (refs. 8,9), we would not have been able to generate iPS cells and advance the field at such rapid speed. In fact, my encounter with mouse ES cells led me to start the ekiden toward iPS cell generation.

Novel caspase‐suicide proteins for tamoxifen‐inducible apoptosis

Taking advantage of a mutant estrogen receptor ligand binding domain (ER(T2)), we developed novel Caspase fusion proteins for inducible apoptosis. We show that Caspase-ER(T2) fusion proteins become specifically activated by the synthetic ligand 4-OH- tamoxifen and rapidly induce apoptotic cell death in human, murine, and zebrafish cells. This novel tool for targeted cell ablation greatly facilitates the generation of disease models as well as developmental and regeneration studies in model organisms.

Expression mapping of tetracycline-responsive prion protein promoter: Digital atlasing for generating cell-specific disease models

We present a digital atlas system that allows mapping of molecular expression patterns at cellular resolution through large series of histological sections. Using this system, we have mapped the distribution of a distinct marker, encoded by the LacZ reporter gene driven by the tetracycline-responsive prion protein promoter in double transgenic mice. The purpose is to evaluate the suitability of this promoter mouse line for targeting genes of interest to specific brain regions, essential for construction of inducible transgenic disease models. Following processing to visualize the promoter expression, sections were counterstained to simultaneously display cytoarchitectonics. High-resolution mosaic images covering entire coronal sections were collected through the mouse brain at intervals of 200 μm. A web-based application provides access to a customized virtual microscopy tool for viewing and navigation within and across the section images. For each section image, the nearest section in a standard atlas is defined, and annotations of key structures and regions inserted. Putative categorization of labeled cells was performed with use of distribution patterns, followed by cell size and shape, as parameters that were compared to legacy data. Among the ensuing results were expression of this promoter in putative glial cells in the cerebellum (and not in Purkinje cells), in putative glial cells in the substantia nigra, in pallidal glial cells or interneurons, and in distinct cell layers and regions of the hippocampus. The study serves as a precursor for a database resource allowing evaluation of the suitability of different promoter mouse lines for generating disease models.

Lentiviral Transgenesis - A Versatile Tool for Basic Research and Gene Therapy

Transgenic animals are of outstanding relevance for medical sciences, because they can be used to model human diseases and to develop gene therapy strategies. A recent development is lentiviral transgenesis: The generation of transgenic animals by lentiviral transduction of oocytes or early embryos. Lentiviral transgenesis is an efficient method to express transgenes in mice and rats as well as in biomedically relevant livestock. Thus, the applications of this technology range from the generation of disease models to gene pharming for human proteins. An important extension of viral transgenesis is the combination of lentiviral gene transfer with RNA interference. Thereby, expression of specific genes can be silenced and loss-of-function models can be generated. Finally, lentiviral transgenic animals can be used to directly evaluate gene therapy strategies that are based on lentiviral vectors prior to their use in humans.

Specific and Potent RNA Interference in Terminally Differentiated Myotubes

Double-stranded RNA (dsRNA) interference is a potent mechanism for sequence-specific silencing of gene expression and represents an invaluable approach for investigating gene function in normal and diseased states as well as for drug target validation. Here, we report that skeletal muscle myoblasts and terminally differentiated myotubes are susceptible to RNA interference. We employed an approach in which dsRNA is generated by cellular transcription from plasmids containing long (1 kilobase) inverted DNA repeats of the target gene rather than using dsRNA synthesized in vitro. We show that gene silencing by this method is effective for endogenously expressed genes as well as for exogenous reporter genes. An analysis of the expression of several endogenous genes and exogenous reporters demonstrates that the silencing effect is specific for the target gene containing sequences within the inverted repeat. Our method eliminates the need to chemically synthesize dsRNA and is not accompanied by global repression of gene expression. Furthermore, we show for the first time that sequence-specific dsRNA-mediated gene silencing is possible in differentiated, multinucleated skeletal muscle myotubes. These findings provide an important molecular tool for the examination of protein function in terminally differentiated muscle cells and provide alternative approaches for generating disease models.

Metaphase II transgenesis

Since its advent in 1974, at least 11 methods have been developed to introduce potentially heritable exogenous DNA (transgenes; tgs) into mammals. These methods are now briefly reviewed in the context of a nascent method that has been demonstrated by microinjection of membrane-depleted sperm heads and tg DNA into metaphase II (mII) oocytes: mII transgenesis. The efficiency of mII transgenesis is at least as high as that of the well-established and prevailing alternative, pronuclear microinjection. Moreover, mII transgenesis promises to facilitate large tg delivery to assist with the generation of disease models and other paradigms in mammalian genome engineering.

Yeast artificial chromosomes (YACs) and the analysis of complex genomes

The development of yeast artificial chromosome (YAC) cloning vectors capable of carrying several hundred kilobase-pairs of DNA insert has greatly facilitated the study of complex genomes, and the cloning of large genes as single fragments. In addition, the ability to manipulate YAC sequences by homologous recombination makes this system extremely useful for the generation of disease models.