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1.
Nat Commun ; 10(1): 2905, 2019 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-31266953

RESUMO

Delivery into mammalian cells remains a significant challenge for many applications of proteins as research tools and therapeutics. We recently reported that the fusion of cargo proteins to a supernegatively charged (-30)GFP enhances encapsulation by cationic lipids and delivery into mammalian cells. To discover polyanionic proteins with optimal delivery properties, we evaluate negatively charged natural human proteins for their ability to deliver proteins into cultured mammalian cells and human primary fibroblasts. Here we discover that ProTα, a small, widely expressed, intrinsically disordered human protein, enables up to ~10-fold more efficient cationic lipid-mediated protein delivery compared to (-30)GFP. ProTα enables efficient delivery at low- to mid-nM concentrations of two unrelated genome editing proteins, Cre recombinase and zinc-finger nucleases, under conditions in which (-30)GFP fusion or cationic lipid alone does not result in substantial activity. ProTα may enable mammalian cell protein delivery applications when delivery potency is limiting.


Assuntos
Edição de Genes/métodos , Lipossomos/química , Proteínas/química , Edição de Genes/instrumentação , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Integrases/química , Integrases/genética , Integrases/metabolismo , Lipossomos/metabolismo , Transporte Proteico , Proteínas/genética , Proteínas/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Nucleases de Dedos de Zinco/química , Nucleases de Dedos de Zinco/genética , Nucleases de Dedos de Zinco/metabolismo
2.
Nature ; 571(7764): 219-225, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31189177

RESUMO

Conventional CRISPR-Cas systems maintain genomic integrity by leveraging guide RNAs for the nuclease-dependent degradation of mobile genetic elements, including plasmids and viruses. Here we describe a notable inversion of this paradigm, in which bacterial Tn7-like transposons have co-opted nuclease-deficient CRISPR-Cas systems to catalyse RNA-guided integration of mobile genetic elements into the genome. Programmable transposition of Vibrio cholerae Tn6677 in Escherichia coli requires CRISPR- and transposon-associated molecular machineries, including a co-complex between the DNA-targeting complex Cascade and the transposition protein TniQ. Integration of donor DNA occurs in one of two possible orientations at a fixed distance downstream of target DNA sequences, and can accommodate variable length genetic payloads. Deep-sequencing experiments reveal highly specific, genome-wide DNA insertion across dozens of unique target sites. This discovery of a fully programmable, RNA-guided integrase lays the foundation for genomic manipulations that obviate the requirements for double-strand breaks and homology-directed repair.


Assuntos
Sistemas CRISPR-Cas/genética , Elementos de DNA Transponíveis/genética , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Edição de Genes/métodos , Mutagênese Insercional/métodos , RNA Bacteriano/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Proteínas Associadas a CRISPR/genética , Proteínas Associadas a CRISPR/metabolismo , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Escherichia coli/genética , Genoma Bacteriano/genética , Integrases/genética , Integrases/metabolismo , Mutagênese Sítio-Dirigida/métodos , RNA Guia/genética , Especificidade por Substrato , Vibrio cholerae/genética
3.
Nat Commun ; 10(1): 2870, 2019 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-31253773

RESUMO

An important channel of cell-to-cell communication is direct contact. The immune synapse is a paradigmatic example of such type of interaction: it forms upon engagement of antigen receptors in lymphocytes by antigen-presenting cells and allows the local exchange of molecules and information. Although mechanics has been shown to play an important role in this process, how forces organize and impact on synapse function is unknown. We find that mechanical forces are spatio-temporally patterned at the immune synapse: global pulsatile myosin II-driven tangential forces are observed at the synapse periphery while localised forces generated by invadosome-like F-actin protrusions are detected at its centre. Noticeably, we observe that these force-producing actin protrusions constitute the main site of antigen extraction and endocytosis and require myosin II contractility to form. The interplay between global and local forces dictated by the organization of the actomyosin cytoskeleton therefore controls endocytosis at the immune synapse.


Assuntos
Citoesqueleto de Actina/fisiologia , Actomiosina/metabolismo , Linfócitos B/fisiologia , Endocitose/fisiologia , Miosina Tipo II/metabolismo , Actomiosina/genética , Animais , Comunicação Celular , Cruzamentos Genéticos , Integrases/genética , Integrases/metabolismo , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Miosina Tipo II/genética , Receptores de Complemento 3d
4.
Nat Protoc ; 14(6): 1820-1840, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31110297

RESUMO

Fate mapping is a powerful genetic tool for linking stem or progenitor cells with their progeny, and hence for defining cell lineages in vivo. The resolution of fate mapping depends on the numbers of distinct markers that are introduced in the beginning into stem or progenitor cells; ideally, numbers should be sufficiently large to allow the tracing of output from individual cells. Highly diverse genetic barcodes can serve this purpose. We recently developed an endogenous genetic barcoding system, termed Polylox. In Polylox, random DNA recombination can be induced by transient activity of Cre recombinase in a 2.1-kb-long artificial recombination substrate that has been introduced into a defined locus in mice (Rosa26Polylox reporter mice). Here, we provide a step-by-step protocol for the use of Polylox, including barcode induction and estimation of induction efficiency, barcode retrieval with single-molecule real-time (SMRT) DNA sequencing followed by computational barcode identification, and the calculation of barcode-generation probabilities, which is key for estimations of single-cell labeling for a given number of stem cells. Thus, Polylox barcoding enables high-resolution fate mapping in essentially all tissues in mice for which inducible Cre driver lines are available. Alternative methods include ex vivo cell barcoding, inducible transposon insertion and CRISPR-Cas9-based barcoding; Polylox currently allows combining non-invasive and cell-type-specific labeling with high label diversity. The execution time of this protocol is ~2-3 weeks for experimental data generation and typically <2 d for computational Polylox decoding and downstream analysis.


Assuntos
Linhagem da Célula , Genes Reporter , Análise de Sequência de DNA/métodos , Animais , Sistemas CRISPR-Cas , DNA/genética , DNA/metabolismo , Código de Barras de DNA Taxonômico/métodos , Feminino , Técnicas de Genotipagem/métodos , Integrases/genética , Masculino , Camundongos , Camundongos Transgênicos , Reação em Cadeia da Polimerase/métodos , Recombinação Genética
5.
Nat Commun ; 10(1): 2262, 2019 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-31118412

RESUMO

Most biomedical research aimed at understanding gene function uses the Cre-Lox system, which consists of the Cre recombinase-dependent deletion of genes containing LoxP sites. This system enables conditional genetic modifications because the expression and activity of the recombinase Cre/CreERT2 can be regulated in space by tissue-specific promoters and in time by the ligand tamoxifen. Since the precise Cre-Lox recombination event is invisible, methods were developed to report Cre activity and are widely used. However, numerous studies have shown that expression of a given Cre activity reporter cannot be assumed to indicate deletion of other LoxP-flanked genes of interest. Here, we report the generation of an inducible dual reporter-Cre mouse allele, iSuRe-Cre. By significantly increasing Cre activity in reporter-expressing cells, iSuRe-Cre provides certainty that these cells have completely recombined floxed alleles. This genetic tool increases the ease, efficiency, and reliability of conditional mutagenesis and gene function analysis.


Assuntos
Edição de Genes/métodos , Vetores Genéticos/genética , Integrases/genética , Plasmídeos/genética , Animais , Técnicas de Cultura de Células , Clonagem Molecular/métodos , Camundongos , Camundongos Transgênicos , Células-Tronco Embrionárias Murinas , Regiões Promotoras Genéticas/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , Recombinação Genética/efeitos dos fármacos , Tamoxifeno/farmacologia
6.
mSphere ; 4(2)2019 04 10.
Artigo em Inglês | MEDLINE | ID: mdl-30971446

RESUMO

Most urinary tract infections (UTIs) are caused by uropathogenic Escherichia coli (UPEC), which depends on an extracellular organelle (type 1 pili) for adherence to bladder cells during infection. Type 1 pilus expression is partially regulated by inversion of a piece of DNA referred to as fimS, which contains the promoter for the fim operon encoding type 1 pili. fimS inversion is regulated by up to five recombinases collectively known as Fim recombinases. These Fim recombinases are currently known to regulate two other switches: the ipuS and hyxS switches. A long-standing question has been whether the Fim recombinases regulate the inversion of other switches, perhaps to coordinate expression for adhesion or virulence. We answered this question using whole-genome sequencing with a newly developed algorithm (structural variation detection using relative entropy [SVRE]) for calling structural variations using paired-end short-read sequencing. SVRE identified all of the previously known switches, refining the specificity of which recombinases act at which switches. Strikingly, we found no new inversions that were mediated by the Fim recombinases. We conclude that the Fim recombinases are each highly specific for a small number of switches. We hypothesize that the unlinked Fim recombinases have been recruited to regulate fimS, and fimS only, as a secondary locus; this further implies that regulation of type 1 pilus expression (and its role in gastrointestinal and/or genitourinary colonization) is important enough, on its own, to influence the evolution and maintenance of multiple additional genes within the accessory genome of E. coli IMPORTANCE UTI is a common ailment that affects more than half of all women during their lifetime. The leading cause of UTIs is UPEC, which relies on type 1 pili to colonize and persist within the bladder during infection. The regulation of type 1 pili is remarkable for an epigenetic mechanism in which a section of DNA containing a promoter is inverted. The inversion mechanism relies on what are thought to be dedicated recombinase genes; however, the full repertoire for these recombinases is not known. We show here that there are no additional targets beyond those already identified for the recombinases in the entire genome of two UPEC strains, arguing that type 1 pilus expression itself is the driving evolutionary force for the presence of these recombinase genes. This further suggests that targeting the type 1 pilus is a rational alternative nonantibiotic strategy for the treatment of UTI.


Assuntos
Proteínas de Fímbrias/genética , Fímbrias Bacterianas/genética , Regulação Bacteriana da Expressão Gênica , Inversão de Sequência , Escherichia coli Uropatogênica/genética , Algoritmos , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Entropia , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Fímbrias/química , Humanos , Integrases/química , Integrases/genética , Regiões Promotoras Genéticas , Infecções Urinárias/microbiologia , Virulência/genética
7.
Nat Commun ; 10(1): 1937, 2019 04 26.
Artigo em Inglês | MEDLINE | ID: mdl-31028261

RESUMO

The development of site-specific recombinases (SSRs) as genome editing agents is limited by the difficulty of altering their native DNA specificities. Here we describe Rec-seq, a method for revealing the DNA specificity determinants and potential off-target substrates of SSRs in a comprehensive and unbiased manner. We applied Rec-seq to characterize the DNA specificity determinants of several natural and evolved SSRs including Cre, evolved variants of Cre, and other SSR family members. Rec-seq profiling of these enzymes and mutants thereof revealed previously uncharacterized SSR interactions, including specificity determinants not evident from SSR:DNA structures. Finally, we used Rec-seq specificity profiles to predict off-target substrates of Tre and Brec1 recombinases, including endogenous human genomic sequences, and confirmed their ability to recombine these off-target sequences in human cells. These findings establish Rec-seq as a high-resolution method for rapidly characterizing the DNA specificity of recombinases with single-nucleotide resolution, and for informing their further development.


Assuntos
DNA Nucleotidiltransferases/genética , DNA/genética , Edição de Genes/métodos , Genoma Humano , Integrases/genética , Sequência de Bases , Clonagem Molecular , DNA/metabolismo , DNA Nucleotidiltransferases/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Células HEK293 , Humanos , Integrases/metabolismo , Oligodesoxirribonucleotídeos/síntese química , Oligodesoxirribonucleotídeos/genética , Oligodesoxirribonucleotídeos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Recombinação Genética
8.
Methods Mol Biol ; 1936: 249-274, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30820904

RESUMO

Cell-type-specific gene targeting with the Cre/loxP system has become an indispensable technique in experimental neuroscience, particularly for the study of late-born glial cells that make myelin. A plethora of conditional mutants and Cre-expressing mouse lines is now available to the research community that allows laboratories to readily engage in in vivo analyses of oligodendrocytes and their precursor cells. This chapter summarizes concepts and strategies in targeting myelinating glial cells in mice for mutagenesis or imaging, and provides an overview of the most important Cre driver lines successfully used in this rapidly growing field.


Assuntos
Marcação de Genes/métodos , Integrases/metabolismo , Oligodendroglia/citologia , Tamoxifeno/farmacologia , Animais , Linhagem da Célula , Regulação da Expressão Gênica/efeitos dos fármacos , Integrases/genética , Camundongos , Camundongos Transgênicos , Mutagênese , Especificidade de Órgãos , Transgenes
9.
Molecules ; 24(6)2019 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-30917606

RESUMO

The genetic modification of the mouse genome using the cre-lox system has been an invaluable tool in deciphering gene and protein function in a temporal and/or spatial manner. However, it has its pitfalls, as researchers have shown that the unregulated expression of cre recombinase can cause DNA damage, the consequences of which can be very detrimental to mouse health. Previously published literature on the most utilized cardiac-specific cre, αMHC-cre, mouse model exhibited a nonlethal hypertrophic cardiomyopathy (HCM) with aging. However, using the same αMHC-cre mice, we observed a cardiac pathology, resulting in complete lethality by 11 months of age. Echocardiography and histology revealed that the αMHC-cre mice were displaying symptoms of dilated cardiomyopathy (DCM) by seven months of age, which ultimately led to their demise in the absence of any HCM at any age. Molecular analysis showed that this phenotype was associated with the DNA damage response through the downregulation of activated p38 and increased expression of JNK, p53, and Bax, known inducers of myocyte death resulting in fibrosis. Our data urges strong caution when interpreting the phenotypic impact of gene responses using αMHC-cre mice, since a lethal DCM was induced by the cre driver in an age-dependent manner in this commonly utilized model system.


Assuntos
Envelhecimento/genética , Cardiomiopatia Dilatada/diagnóstico por imagem , Integrases/metabolismo , Cadeias Pesadas de Miosina/genética , Envelhecimento/metabolismo , Animais , Cardiomiopatia Dilatada/genética , Cardiomiopatia Dilatada/metabolismo , Dano ao DNA , Modelos Animais de Doenças , Ecocardiografia , Regulação da Expressão Gênica , Genes Letais , Integrases/genética , Camundongos , Cadeias Pesadas de Miosina/metabolismo , Fenótipo
10.
Mol Cell ; 73(4): 727-737.e3, 2019 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-30709710

RESUMO

CRISPR-Cas immunity requires integration of short, foreign DNA fragments into the host genome at the CRISPR locus, a site consisting of alternating repeat sequences and foreign-derived spacers. In most CRISPR systems, the proteins Cas1 and Cas2 form the integration complex and are both essential for DNA acquisition. Most type V-C and V-D systems lack the cas2 gene and have unusually short CRISPR repeats and spacers. Here, we show that a mini-integrase comprising the type V-C Cas1 protein alone catalyzes DNA integration with a preference for short (17- to 19-base-pair) DNA fragments. The mini-integrase has weak specificity for the CRISPR array. We present evidence that the Cas1 proteins form a tetramer for integration. Our findings support a model of a minimal integrase with an internal ruler mechanism that favors shorter repeats and spacers. This minimal integrase may represent the function of the ancestral Cas1 prior to Cas2 adoption.


Assuntos
Proteínas Associadas a CRISPR/genética , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , DNA Bacteriano/genética , Endodesoxirribonucleases/genética , Endonucleases/genética , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Edição de Genes/métodos , Integrases/genética , Pareamento de Bases , Proteínas Associadas a CRISPR/metabolismo , DNA Bacteriano/metabolismo , Endodesoxirribonucleases/metabolismo , Endonucleases/metabolismo , Escherichia coli/enzimologia , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Integrases/metabolismo , Motivos de Nucleotídeos , Especificidade por Substrato
11.
Hum Genomics ; 13(1): 10, 2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30770771

RESUMO

BACKGROUND: Despite a number of different transgenes that can mediate DNA deletion in the developing lens, each has unique features that can make a given transgenic line more or less appropriate for particular studies. The purpose of this work encompasses both a review of transgenes that lead to the expression of Cre recombinase in the lens and a comparative analysis of currently available transgenic lines with a particular emphasis on the Le-Cre and P0-3.9GFPCre lines that can mediate DNA deletion in the lens placode. Although both of these transgenes are driven by elements of the Pax6 P0 promoter, the Le-Cre transgene consistently leads to ocular abnormalities in homozygous state and can lead to ocular defects on some genetic backgrounds when hemizygous. RESULT: Although both P0-3.9GFPCre and Le-Cre hemizygous transgenic mice undergo normal eye development on an FVB/N genetic background, Le-Cre homozygotes uniquely exhibit microphthalmia. Examination of the expression patterns of these two transgenes revealed similar expression in the developing eye and pancreas. However, lineage tracing revealed widespread non-ocular CRE reporter gene expression in the P0-3.9GFPCre transgenic mice that results from stochastic CRE expression in the P0-3.9GFPCre embryos prior to lens placode formation. Postnatal hemizygous Le-Cre transgenic lenses express higher levels of CRE transcript and protein than the hemizygous lenses of P0-3.9GFPCre mice. Transcriptome analysis revealed that Le-Cre hemizygous lenses deregulated the expression of 15 murine genes, several of which are associated with apoptosis. In contrast, P0-3.9GFPCre hemizygous lenses only deregulated two murine genes. No known PAX6-responsive genes or genes directly associated with lens differentiation were deregulated in the hemizygous Le-Cre lenses. CONCLUSIONS: Although P0-3.9GFPCre transgenic mice appear free from ocular abnormalities, extensive non-ocular CRE expression represents a potential problem for conditional gene deletion studies using this transgene. The higher level of CRE expression in Le-Cre lenses versus P0-3.9GFPCre lenses may explain abnormal lens development in homozygous Le-Cre mice. Given the lack of deregulation of PAX6-responsive transcripts, we suggest that abnormal eye development in Le-Cre transgenic mice stems from CRE toxicity. Our studies reinforce the requirement for appropriate CRE-only expressing controls when using CRE as a driver of conditional gene targeting strategies.


Assuntos
Deleção de Genes , Integrases/genética , Cristalino/fisiologia , Camundongos Transgênicos , Animais , Feminino , Regulação da Expressão Gênica , Proteínas de Fluorescência Verde/genética , Cristalino/embriologia , Cristalino/fisiopatologia , Camundongos Endogâmicos
12.
Lett Appl Microbiol ; 68(5): 370-377, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30790321

RESUMO

Aeromonas spp. are Gram-negative opportunistic bacteria which have been commonly associated with fish diseases. In this study, antibiogram, antimicrobial resistance genes and integrons of 43 zebrafish-borne Aeromonas spp. were studied. The isolates were identified as six Aeromonas species (A. veronii biovar veronii (n = 26), A. veronii biovar sobria (n = 3), A. hydrophila (n = 8), A. caviae (n = 3), A. enteropelogenes (n = 2) and A. dhakensis (n = 1)). Antibiogram of the isolates indicated that most of them were resistant to amoxicillin (100·00%), nalidixic acid (100·00%), oxytetracycline (100·00%), ampicillin (93·02%), tetracycline (74·42%), rifampicin (67·44%) and imipenem (65·15%). Multiple antimicrobial resistance (MAR) index values ranged from 0·19-0·44 to 90·70% isolates showed multidrug resistance. PCR of antimicrobial resistance genes revealed that the tetracycline resistance gene (tetA) was the most predominant (67·44%) among the isolates. The qnrS (53·49%), tetB (30·23%), tetE (30·23%), qnrB (23·26%) and aac(6')-Ib-cr (4·65%) genes were also detected. Class 1 integrase (IntI1) gene was found in 46·51% of the isolates. Two types of class 1 integron gene cassette profiles (qacG-aadA6-qacG and drfA1) were identified. The results showed that zebrafish-borne aeromonads can harbour different types of antimicrobial resistance genes and class 1 integrons. SIGNIFICANCE AND IMPACT OF THE STUDY: Aeromonas spp. are important pathogens found in diverse environments. Antimicrobial resistance genes and integrons of ornamental fish-borne Aeromonas spp. are not well studied. The antibiogram, antimicrobial resistance genes and class 1 integrons of Aeromonas spp. isolated from zebrafish were characterized for the first time in Korea. The prevalence of tetracycline resistance genes, plasmid-mediated quinolone resistance genes and class 1 integron gene cassettes were observed among the isolates. The qacG-aadA6-qacG gene cassette was identified for the first time in Aeromonas spp. The results suggest that the wise use of antimicrobials is necessary for the better management of the ornamental fish.


Assuntos
Aeromonas/genética , Antibacterianos/farmacologia , Farmacorresistência Bacteriana Múltipla/genética , Integrases/genética , Integrons/genética , Aeromonas/classificação , Aeromonas/efeitos dos fármacos , Animais , Doenças dos Peixes/microbiologia , Testes de Sensibilidade Microbiana , Plasmídeos/genética , Reação em Cadeia da Polimerase , República da Coreia , Peixe-Zebra/microbiologia
13.
Methods Mol Biol ; 1929: 223-231, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30710276

RESUMO

The calcineurin/nuclear factor of activated T-cell (NFAT) signaling pathway is important for the development and function of different leukocyte subsets. Calcineurin is activated through increased intracellular levels of calcium that is triggered by cell receptors. The phosphatase activity of calcineurin dephosporylates the members of NFAT transcription factor family and activates their translocation to the nucleus and further starts the transcription of NFAT-regulated genes. Although calcineurin is expressed ubiquitously, its functions are cell- and tissue-specific. The genetically engineered murine models allow to dissect the calcineurin roles in tissue-specific manner and as such have been essential to pursue the understanding of important role of calcineurin/NFAT pathway in orchestrating the developmental and immune response processes. This protocol describes several examples of the use of Cre/loxP inducible mouse models deficient in calcineurin in different myeloid subsets to dissect the cell- and tissue-specific roles of calcineurin signaling.


Assuntos
Calcineurina/genética , Integrases/genética , Células Mieloides/metabolismo , Transdução de Sinais , Animais , Calcineurina/metabolismo , Cálcio/metabolismo , Núcleo Celular/metabolismo , Células Cultivadas , Técnicas de Inativação de Genes , Camundongos
14.
Methods Mol Biol ; 1926: 3-21, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30742258

RESUMO

For studies of gene function during development, it can be very useful to generate mosaic embryos in which a small subset of cells in a given cell lineage lacks a gene of interest and carries a marker that allows the mutant cells to be specifically visualized and compared to wild-type cells. Several methods have been used to generate genetically mosaic mouse kidneys for such studies. These include (1) chimeric embryos generated using embryonic stem cells, (2) chimeric renal organoids generated by dissociation and reaggregation of the fetal kidneys, (3) generation of a knockout allele with a built-in reporter gene, (4) mosaic analysis with double markers (MADM), and (5) mosaic mutant analysis with spatial and temporal control of recombination (MASTR). In this chapter, these five methods are described, and their advantages and disadvantages are discussed.


Assuntos
Rim/citologia , Rim/embriologia , Organoides/citologia , Animais , DNA Nucleotidiltransferases/genética , DNA Nucleotidiltransferases/metabolismo , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Integrases/genética , Integrases/metabolismo , Rim/metabolismo , Camundongos , Camundongos Knockout , Organoides/metabolismo
15.
Genetics ; 211(4): 1155-1177, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30765420

RESUMO

To understand gene function, the cre/loxP conditional system is the most powerful available for temporal and spatial control of expression in mouse. However, the research community requires more cre recombinase expressing transgenic mouse strains (cre-drivers) that restrict expression to specific cell types. To address these problems, a high-throughput method for large-scale production that produces high-quality results is necessary. Further, endogenous promoters need to be chosen that drive cell type specific expression, or we need to further focus the expression by manipulating the promoter. Here we test the suitability of using knock-ins at the docking site 5' of Hprt for rapid development of numerous cre-driver strains focused on expression in adulthood, using an improved cre tamoxifen inducible allele (icre/ERT2), and testing a novel inducible-first, constitutive-ready allele (icre/f3/ERT2/f3). In addition, we test two types of promoters either to capture an endogenous expression pattern (MaxiPromoters), or to restrict expression further using minimal promoter element(s) designed for expression in restricted cell types (MiniPromoters). We provide new cre-driver mouse strains with applicability for brain and eye research. In addition, we demonstrate the feasibility and applicability of using the locus 5' of Hprt for the rapid generation of substantial numbers of cre-driver strains. We also provide a new inducible-first constitutive-ready allele to further speed cre-driver generation. Finally, all these strains are available to the research community through The Jackson Laboratory.


Assuntos
Encéfalo/metabolismo , Olho/metabolismo , Técnicas de Introdução de Genes/métodos , Camundongos Transgênicos/genética , Tamoxifeno/farmacologia , Ativação Transcricional/efeitos dos fármacos , Animais , Efeito Fundador , Hipoxantina Fosforribosiltransferase/genética , Hipoxantina Fosforribosiltransferase/metabolismo , Integrases/genética , Integrases/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Regiões Promotoras Genéticas
16.
Brain ; 142(2): 362-375, 2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30601941

RESUMO

De novo mutations of the sodium channel gene SCN8A result in an epileptic encephalopathy with refractory seizures, developmental delay, and elevated risk of sudden death. p.Arg1872Trp is a recurrent de novo SCN8A mutation reported in 14 unrelated individuals with epileptic encephalopathy that included seizure onset in the prenatal or infantile period and severe verbal and ambulatory comorbidities. The major biophysical effect of the mutation was previously shown to be impaired channel inactivation accompanied by increased current density. We have generated a conditional mouse mutation in which expression of this severe gain-of-function mutation is dependent upon Cre recombinase. Global activation of p.Arg1872Trp by EIIa-Cre resulted in convulsive seizures and lethality at 2 weeks of age. Neural activation of the p.Arg1872Trp mutation by Nestin-Cre also resulted in early onset seizures and death. Restriction of p.Arg1872Trp expression to excitatory neurons using Emx1-Cre recapitulated seizures and juvenile lethality between 1 and 2 months of age. In contrast, activation of p.Arg1872Trp in inhibitory neurons by Gad2-Cre or Dlx5/6-Cre did not induce seizures or overt neurological dysfunction. The sodium channel modulator GS967/Prax330 prolonged survival of mice with global expression of R1872W and also modulated the activity of the mutant channel in transfected cells. Activation of the p.Arg1872Trp mutation in adult mice was sufficient to generate seizures and death, indicating that successful therapy will require lifelong treatment. These findings provide insight into the pathogenic mechanism of this gain-of-function mutation of SCN8A and identify excitatory neurons as critical targets for therapeutic intervention.


Assuntos
Encefalopatias/genética , Potenciais Pós-Sinápticos Excitadores/fisiologia , Integrases/genética , Canal de Sódio Disparado por Voltagem NAV1.6/genética , Neurônios/fisiologia , Prosencéfalo/fisiologia , Animais , Encefalopatias/patologia , Células Cultivadas , Feminino , Mutação com Ganho de Função/genética , Integrases/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neurônios/patologia , Técnicas de Cultura de Órgãos , Prosencéfalo/patologia
17.
Microb Genom ; 5(2)2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30672731

RESUMO

Avian pathogenic Escherichia coli (APEC) cause widespread economic losses in poultry production and are potential zoonotic pathogens. Genome sequences of 95 APEC from commercial poultry operations in four Australian states that carried the class 1 integrase gene intI1, a proxy for multiple drug resistance (MDR), were characterized. Sequence types ST117 (22/95), ST350 (10/95), ST429 and ST57 (each 9/95), ST95 (8/95) and ST973 (7/95) dominated, while 24 STs were represented by one or two strains. FII and FIB repA genes were the predominant (each 93/95, 98 %) plasmid incompatibility groups identified, but those of B/O/K/Z (25/95, 26 %) and I1 (24/95, 25 %) were also identified frequently. Virulence-associated genes (VAGs) carried by ColV and ColBM virulence plasmids, including those encoding protectins [iss (91/95, 96 %), ompT (91/95, 96 %) and traT (90/95, 95 %)], iron-acquisition systems [sitA (88/95, 93 %), etsA (87/95, 92 %), iroN (84/95, 89 %) and iucD/iutA (84/95, 89 %)] and the putative avian haemolysin hylF (91/95, 96 %), featured prominently. Notably, mobile resistance genes conferring resistance to fluoroquinolones, colistin, extended-spectrum ß-lactams and carbapenems were not detected in the genomes of these 95 APEC but carriage of the sulphonamide resistance gene, sul1 (59/95, 63 %), the trimethoprim resistance gene cassettes dfrA5 (48/95, 50 %) and dfrA1 (25/95, 27 %), the tetracycline resistance determinant tet(A) (51/95, 55 %) and the ampicillin resistance genes blaTEM-1A/B/C (48/95, 52 %) was common. IS26 (77/95, 81 %), an insertion element known to capture and mobilize a wide spectrum of antimicrobial resistance genes, was also frequently identified. These studies provide a baseline snapshot of drug-resistant APEC in Australia and their role in the carriage of ColV-like virulence plasmids.


Assuntos
Farmacorresistência Bacteriana Múltipla/genética , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/veterinária , Escherichia coli/classificação , Escherichia coli/genética , Doenças das Aves Domésticas/microbiologia , Animais , Austrália , Toxinas Bacterianas/genética , Elementos de DNA Transponíveis , DNA Bacteriano/genética , Escherichia coli/patogenicidade , Proteínas de Escherichia coli/genética , Genoma Bacteriano , Integrases/genética , Plasmídeos , Análise de Sequência de DNA/métodos , Virulência/genética , Fatores de Virulência/genética , Sequenciamento Completo do Genoma
18.
Nat Neurosci ; 22(3): 492-502, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30692688

RESUMO

Functionally and anatomically distinct cortical substructures, such as areas or layers, contain different principal neuron (PN) subtypes that generate output signals representing particular information. Various types of cortical inhibitory interneurons (INs) differentially but coordinately regulate PN activity. Despite a potential determinant for functional specialization of PN subtypes, the spatial organization of IN subtypes that innervate defined PN subtypes remains unknown. Here we develop a genetic strategy combining a recombinase-based intersectional labeling method and rabies viral monosynaptic tracing, which enables subtype-specific visualization of cortical IN ensembles sending inputs to defined PN subtypes. Our approach reveals not only cardinal but also underrepresented connections between broad, non-overlapping IN subtypes and PNs. Furthermore, we demonstrate that distinct PN subtypes defined by areal or laminar positions display different organization of input IN subtypes. Our genetic strategy will facilitate understanding of the wiring and developmental principles of cortical inhibitory circuits at unparalleled levels.


Assuntos
Córtex Cerebral/fisiologia , Neurônios GABAérgicos/fisiologia , Interneurônios/fisiologia , Técnicas de Rastreamento Neuroanatômico/métodos , Sinapses/fisiologia , Potenciais de Ação , Animais , Axônios , Córtex Cerebral/citologia , Feminino , Neurônios GABAérgicos/citologia , Vetores Genéticos , Integrases/genética , Interneurônios/citologia , Masculino , Camundongos Transgênicos , Vias Neurais/citologia , Vias Neurais/fisiologia , Neurônios/citologia , Neurônios/fisiologia , Vírus da Raiva/genética
19.
Nature ; 566(7742): 105-109, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30675057

RESUMO

A gene drive biases the transmission of one of the two copies of a gene such that it is inherited more frequently than by random segregation. Highly efficient gene drive systems have recently been developed in insects, which leverage the sequence-targeted DNA cleavage activity of CRISPR-Cas9 and endogenous homology-directed repair mechanisms to convert heterozygous genotypes to homozygosity1-4. If implemented in laboratory rodents, similar systems would enable the rapid assembly of currently impractical genotypes that involve multiple homozygous genes (for example, to model multigenic human diseases). To our knowledge, however, such a system has not yet been demonstrated in mammals. Here we use an active genetic element that encodes a guide RNA, which is embedded in the mouse tyrosinase (Tyr) gene, to evaluate whether targeted gene conversion can occur when CRISPR-Cas9 is active in the early embryo or in the developing germline. Although Cas9 efficiently induces double-stranded DNA breaks in the early embryo and male germline, these breaks are not corrected by homology-directed repair. By contrast, Cas9 expression limited to the female germline induces double-stranded breaks that are corrected by homology-directed repair, which copies the active genetic element from the donor to the receiver chromosome and increases its rate of inheritance in the next generation. These results demonstrate the feasibility of CRISPR-Cas9-mediated systems that bias inheritance of desired alleles in mice and that have the potential to transform the use of rodent models in basic and biomedical research.


Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Conversão Gênica , Tecnologia de Impulso Genético/métodos , Mutação em Linhagem Germinativa/genética , Heterozigoto , Homozigoto , Alelos , Animais , Cruzamento , Proteína 9 Associada à CRISPR/genética , Cromossomos de Mamíferos/genética , Quebras de DNA de Cadeia Dupla , Modelos Animais de Doenças , Embrião de Mamíferos/enzimologia , Embrião de Mamíferos/metabolismo , Feminino , Integrases/genética , Integrases/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Monofenol Mono-Oxigenase/genética , RNA Guia/genética , Transgenes/genética
20.
Nat Protoc ; 14(2): 556-575, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30610240

RESUMO

A big challenge in proteomics is the identification of cell-type-specific proteomes in vivo. This protocol describes how to label, purify and identify cell-type-specific proteomes in living mice. To make this possible, we created a Cre-recombinase-inducible mouse line expressing a mutant methionyl-tRNA synthetase (L274G), which enables the labeling of nascent proteins with the non-canonical amino acid azidonorleucine (ANL). This amino acid can be conjugated to different affinity tags by click chemistry. After affinity purification (AP), the labeled proteins can be identified by tandem mass spectrometry (MS/MS). With this method, it is possible to identify cell-type-specific proteomes derived from living animals, which was not possible with any previously published method. The reduction in sample complexity achieved by this protocol allows for the detection of subtle changes in cell-type-specific protein content in response to environmental changes. This protocol can be completed in ~10 d (plus the time needed to generate the mouse lines, the desired labeling period and MS analysis).


Assuntos
Azidas/metabolismo , Química Click/métodos , Metionina tRNA Ligase/genética , Norleucina/análogos & derivados , Proteoma/isolamento & purificação , Proteômica/métodos , Coloração e Rotulagem/métodos , Animais , Expressão Gênica , Integrases/genética , Integrases/metabolismo , Metionina tRNA Ligase/metabolismo , Camundongos , Camundongos Transgênicos , Mutação , Norleucina/metabolismo , Especificidade de Órgãos , Proteoma/biossíntese , Proteoma/genética , Purificação por Afinidade em Tandem/métodos , Espectrometria de Massas em Tandem
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