RESUMO
CRISPR-based transcriptional activation is a powerful tool for functional gene interrogation; however, delivery difficulties have limited its applications in vivo. Here, we created a mouse model expressing all components of the CRISPR-Cas9 guide RNA-directed Synergistic Activation Mediator (SAM) from a single transcript that is capable of activating target genes in a tissue-specific manner. We optimized Lipid Nanoparticles and Adeno-Associated Virus guide RNA delivery approaches to achieve expression modulation of one or more genes in vivo. We utilized the SAM mouse model to generate a hypercholesteremia disease state that we could bidirectionally modulate with various guide RNAs. Additionally, we applied SAM to optimize gene expression in a humanized Transthyretin mouse model to recapitulate human expression levels. These results demonstrate that the SAM gene activation platform can facilitate in vivo research and drug discovery.
Assuntos
Sistemas CRISPR-Cas/genética , Hipercolesterolemia/genética , Lipossomos/farmacologia , Pré-Albumina/metabolismo , Ativação Transcricional/genética , Animais , Linhagem Celular , Expressão Gênica/genética , Regulação da Expressão Gênica/genética , Engenharia Genética/métodos , Células HEK293 , Humanos , Hipercolesterolemia/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Nanopartículas , Pré-Albumina/genética , RNA Guia de Cinetoplastídeos/genética , RNA Guia de Cinetoplastídeos/metabolismoRESUMO
INTRODUCTION: Latent TGFß binding proteins (LTBPs) govern TGFß presentation and activation and are important for elastogenesis. Although TGFß is well-known as a tumor suppressor and metastasis promoter, and LTBP1 is elevated in two distinct breast cancer metastasis signatures, LTBPs have not been studied in the normal mammary gland. METHODS: To address this we have examined Ltbp1 promoter activity throughout mammary development using an Ltbp1L-LacZ reporter as well as expression of both Ltbp1L and 1S mRNA and protein by qRT-PCR, immunofluorescence and flow cytometry. RESULTS: Our data show that Ltbp1L is transcribed coincident with lumen formation, providing a rare marker distinguishing ductal from alveolar luminal lineages. Ltbp1L and Ltbp1S are silent during lactation but robustly induced during involution, peaking at the stage when the remodeling process becomes irreversible. Ltbp1L is also induced within the embryonic mammary mesenchyme and maintained within nipple smooth muscle cells and myofibroblasts. Ltbp1 protein exclusively ensheaths ducts and side branches. CONCLUSIONS: These data show Ltbp1 is transcriptionally regulated in a dynamic manner that is likely to impose significant spatial restriction on TGFß bioavailability during mammary development. We hypothesize that Ltbp1 functions in a mechanosensory capacity to establish and maintain ductal luminal cell fate, support and detect ductal distension, trigger irreversible involution, and facilitate nipple sphincter function.
Assuntos
Proteínas de Ligação a TGF-beta Latente/metabolismo , Glândulas Mamárias Animais/citologia , Glândulas Mamárias Animais/embriologia , Mesoderma/citologia , Animais , Linhagem da Célula , Movimento Celular , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Lactação , Proteínas de Ligação a TGF-beta Latente/genética , Glândulas Mamárias Animais/fisiologia , Mesoderma/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Músculo Liso/citologia , Músculo Liso/embriologia , Gravidez , Regiões Promotoras Genéticas , Regulação para CimaRESUMO
MicroRNAs are small RNA species involved in biological control at multiple levels. Using genetic deletion and transgenic approaches, we show that the evolutionarily conserved microRNA-155 (miR-155) has an important role in the mammalian immune system, specifically in regulating T helper cell differentiation and the germinal center reaction to produce an optimal T cell-dependent antibody response. miR-155 exerts this control, at least in part, by regulating cytokine production. These results also suggest that individual microRNAs can exert critical control over mammalian differentiation processes in vivo.
Assuntos
Linfócitos B/imunologia , Centro Germinativo/imunologia , MicroRNAs/fisiologia , Linfócitos T/imunologia , Animais , Diferenciação Celular , Células Cultivadas , Citocinas/biossíntese , Imunoglobulina G/análise , Ativação Linfocitária , Linfotoxina-alfa/biossíntese , Linfotoxina-beta/biossíntese , Camundongos , Camundongos Knockout , Camundongos Transgênicos , MicroRNAs/genética , Nitrofenóis/imunologia , Nódulos Linfáticos Agregados/imunologia , Fenilacetatos , Hipermutação Somática de Imunoglobulina , Baço/imunologia , Linfócitos T/citologia , Linfócitos T/metabolismo , Células Th1/citologia , Células Th1/imunologia , Células Th2/citologia , Células Th2/imunologia , Fator de Necrose Tumoral alfa/biossínteseRESUMO
A useful approach for exploring gene function involves generating mutant mice from genetically modified embryonic stem (ES) cells. Recent advances in genetic engineering of ES cells have shifted the bottleneck in this process to the generation of mice. Conventional injections of ES cells into blastocyst hosts produce F0 generation chimeras that are only partially derived from ES cells, requiring additional breeding to obtain mutant mice that can be phenotyped. The tetraploid complementation approach directly yields mice that are almost entirely derived from ES cells, but it is inefficient, works only with certain hybrid ES cell lines and suffers from nonspecific lethality and abnormalities, complicating phenotypic analyses. Here we show that laser-assisted injection of either inbred or hybrid ES cells into eight cell-stage embryos efficiently yields F0 generation mice that are fully ES cell-derived and healthy, exhibit 100% germline transmission and allow immediate phenotypic analysis, greatly accelerating gene function assignment.
Assuntos
Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/transplante , Marcação de Genes/métodos , Terapia a Laser/métodos , Camundongos Transgênicos/genética , Microinjeções/métodos , Transplante de Células-Tronco/métodos , Animais , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Camundongos Transgênicos/anatomia & histologia , Camundongos Transgênicos/cirurgia , Microcirurgia/métodos , FenótipoRESUMO
The Sir2 histone deacetylase functions as a chromatin silencer to regulate recombination, genomic stability, and aging in budding yeast. Seven mammalian Sir2 homologs have been identified (SIRT1-SIRT7), and it has been speculated that some may have similar functions to Sir2. Here, we demonstrate that SIRT6 is a nuclear, chromatin-associated protein that promotes resistance to DNA damage and suppresses genomic instability in mouse cells, in association with a role in base excision repair (BER). SIRT6-deficient mice are small and at 2-3 weeks of age develop abnormalities that include profound lymphopenia, loss of subcutaneous fat, lordokyphosis, and severe metabolic defects, eventually dying at about 4 weeks. We conclude that one function of SIRT6 is to promote normal DNA repair, and that SIRT6 loss leads to abnormalities in mice that overlap with aging-associated degenerative processes.