ABSTRACT
Systemic Lupus Erythematosus (SLE) is a chronic autoimmune disease representing a serious unmet medical need. The disease is associated with the loss of self-tolerance and exaggerated B cell activation, resulting in autoantibody production and the formation of immune complexes that accumulate in the kidney, causing glomerulonephritis. TLR7, an important mediator of the innate immune response, drives the expression of type-1 interferon (IFN), which leads to expression of type-1 IFN induced genes and aggravates lupus pathology. Because the lysosomal peptide symporter slc15a4 is critically required for type-1 interferon production by pDC, and for certain B cell functions in response to TLR7 and TLR9 signals, we considered it as a potential target for pharmacological intervention in SLE. We deleted the slc15a4 gene in C57BL/6, NZB, and NZW mice and found that pristane-challenged slc15a4-/- mice in the C57BL/6 background and lupus prone slc15a4-/- NZB/W F1 mice were both completely protected from lupus like disease. In the NZB/W F1 model, protection persisted even when disease development was accelerated with an adenovirus encoding IFNα, emphasizing a broad role of slc15a4 in disease initiation. Our results establish a non-redundant function of slc15a4 in regulating both innate and adaptive components of the immune response in SLE pathobiology and suggest that it may be an attractive drug target.
Subject(s)
Lupus Erythematosus, Systemic/pathology , Membrane Transport Proteins/metabolism , Animals , Chemokines/metabolism , Cytokines/metabolism , Dendritic Cells/cytology , Dendritic Cells/drug effects , Dendritic Cells/metabolism , Imidazoles/pharmacology , Interferon-alpha/genetics , Interferon-alpha/metabolism , Interferon-alpha/pharmacology , Lupus Erythematosus, Systemic/metabolism , Lupus Erythematosus, Systemic/mortality , Membrane Transport Proteins/deficiency , Membrane Transport Proteins/genetics , Mice , Mice, Inbred C57BL , Mice, Inbred NZB , Mice, Knockout , Survival Rate , Terpenes/pharmacology , Toll-Like Receptor 7/agonists , Toll-Like Receptor 7/metabolism , Toll-Like Receptor 9/metabolismABSTRACT
Since the first knockout rat model was generated with zinc-finger nucleases (ZFNs) by Geurt's group in 2009, the demand for making targeted rat models has increased tremendously. The advent of the clustered regularly interspaced short palindromic repeats-CRISPR associated protein 9 (CRISPR-Cas9) system provides researchers with a more efficient method for producing modified animals, which has since then been developed and applied in rat. Since we established a rat model production system at our facility in 2014, we have consistently generated rat models. Due to differences in physiology and embryology between mouse and rat, species-specific protocols for superovulation conditions, microinjection, and embryo transfer (among others) are required. There are over 100 rat strains, and Sprague Dawley is one of the commonly used outbred strains in biomedical research. In this chapter, we describe in detail a range of topics including donor and recipient preparation, microinjection setup, CRISPR reagent preparation, and oviduct transfer procedures for making rat models in the Sprague Dawley background.
Subject(s)
CRISPR-Cas Systems , Microinjections/methods , Models, Animal , Oviducts/metabolism , Animals , Cell Nucleus/genetics , Cytoplasm/genetics , Embryo Transfer , Female , Gene Editing , Gene Knockout Techniques , Rats , Rats, Sprague-Dawley , Species Specificity , SuperovulationABSTRACT
Embryonic stem (ES) cells from a C57BL/6N (B6N) background injected into B6(Cg)-Tyrc-2J/J (B6-albino) recipient blastocysts are commonly used for generating genetically modified mouse models. To understand the influence of the recipient blastocyst strain on germline transmission, BALB/cAnNTac and B6-albino germline transmission rates were compared using the C57BL6/N-derived C2 ES cell line. A total of 92 ES cell clones from 27 constructs were injected. We compared blastocyst yield, birth rate, chimera formation rate, and high-percentage (>50 %) male chimera formation rate. For germline transmission, we analyzed 24 clones from 19 constructs, which generated high-percentage male chimeras from both donor strains. B6-albino hosts resulted in higher mean blastocyst yields per donor than did BALB/c ones (3.6 vs. 2.5). However, BALB/c hosts resulted in a higher birth rate than B6-albino ones (36 vs. 27 %), a higher chimera formation rate (50 vs. 42 %), a higher high-percentage male chimera rate (10 vs. 8 %), and a higher germline transmission rate (65 vs. 49 %), respectively. Our data suggest that BALB/c is a suitable blastocyst host strain for C2 ES cells and has an advantage over the B6-albino strain for receiving the injection of C2 ES cells.
Subject(s)
Blastocyst/physiology , Embryonic Stem Cells , Mice, Inbred BALB C , Mice, Inbred C57BL , Animals , Female , Germ Cells , Male , Mice, Transgenic , MicroinjectionsABSTRACT
Dissipating excess calories as heat through therapeutic stimulation of brown adipose tissues (BAT) has been proposed as a potential treatment for obesity-linked disorders. Here, we describe the generation of a humanized effector-less bispecific antibody that activates fibroblast growth factor receptor (FGFR) 1/ßKlotho complex, a common receptor for FGF21 and FGF19. Using this molecule, we show that antibody-mediated activation of FGFR1/ßKlotho complex in mice induces sustained energy expenditure in BAT, browning of white adipose tissue, weight loss, and improvements in obesity-associated metabolic derangements including insulin resistance, hyperglycemia, dyslipidemia and hepatosteatosis. In mice and cynomolgus monkeys, FGFR1/ßKlotho activation increased serum high-molecular-weight adiponectin, which appears to contribute over time by enhancing the amplitude of the metabolic benefits. At the same time, insulin sensitization by FGFR1/ßKlotho activation occurs even before the onset of weight loss in a manner that is independent of adiponectin. Together, selective activation of FGFR1/ßKlotho complex with a long acting therapeutic antibody represents an attractive approach for the treatment of type 2 diabetes and other obesity-linked disorders through enhanced energy expenditure, insulin sensitization and induction of high-molecular-weight adiponectin.
Subject(s)
Adipose Tissue, Brown/metabolism , Antibodies, Bispecific/pharmacology , Insulin/pharmacology , Membrane Proteins/agonists , Receptor, Fibroblast Growth Factor, Type 1/agonists , Adiponectin/metabolism , Adipose Tissue, Brown/drug effects , Animals , Cell Line , Energy Metabolism/drug effects , Fibroblast Growth Factors/pharmacology , HEK293 Cells , Humans , Klotho Proteins , Macaca fascicularis , Male , Membrane Proteins/metabolism , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Obese , Protein Binding/drug effects , Receptor, Fibroblast Growth Factor, Type 1/metabolism , Thermogenesis/drug effects , Weight Loss/drug effectsABSTRACT
We report the characterization of a highly germline competent C57BL/6N mouse embryonic stem cell line, JM8. To simplify breeding schemes, the dominant agouti coat color gene was restored in JM8 cells by targeted repair of the C57BL/6 nonagouti mutation. These cells provide a robust foundation for large-scale mouse knockout programs that aim to provide a public resource of targeted mutations in the C57BL/6 genetic background.
