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1.
Nat Cell Biol ; 22(3): 321-331, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32123335

RESUMO

CRISPR-Cas9 technology has revolutionized genome editing and is applicable to the organoid field. However, precise integration of exogenous DNA sequences into human organoids is lacking robust knock-in approaches. Here, we describe CRISPR-Cas9-mediated homology-independent organoid transgenesis (CRISPR-HOT), which enables efficient generation of knock-in human organoids representing different tissues. CRISPR-HOT avoids extensive cloning and outperforms homology directed repair (HDR) in achieving precise integration of exogenous DNA sequences into desired loci, without the necessity to inactivate TP53 in untransformed cells, which was previously used to increase HDR-mediated knock-in. CRISPR-HOT was used to fluorescently tag and visualize subcellular structural molecules and to generate reporter lines for rare intestinal cell types. A double reporter-in which the mitotic spindle was labelled by endogenously tagged tubulin and the cell membrane by endogenously tagged E-cadherin-uncovered modes of human hepatocyte division. Combining tubulin tagging with TP53 knock-out revealed that TP53 is involved in controlling hepatocyte ploidy and mitotic spindle fidelity. CRISPR-HOT simplifies genome editing in human organoids.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Técnicas de Introdução de Genes/métodos , Organoides/citologia , Hepatócitos/citologia , Hepatócitos/ultraestrutura , Humanos , Intestinos/citologia , Fígado/citologia , Organoides/ultraestrutura , Fuso Acromático/ultraestrutura , Proteína Supressora de Tumor p53/fisiologia
2.
Nat Commun ; 11(1): 1178, 2020 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-32132530

RESUMO

Targeted insertion of transgenes at pre-determined plant genomic safe harbors provides a desirable alternative to insertions at random sites achieved through conventional methods. Most existing cases of targeted gene insertion in plants have either relied on the presence of a selectable marker gene in the insertion cassette or occurred at low frequency with relatively small DNA fragments (<1.8 kb). Here, we report the use of an optimized CRISPR-Cas9-based method to achieve the targeted insertion of a 5.2 kb carotenoid biosynthesis cassette at two genomic safe harbors in rice. We obtain marker-free rice plants with high carotenoid content in the seeds and no detectable penalty in morphology or yield. Whole-genome sequencing reveals the absence of off-target mutations by Cas9 in the engineered plants. These results demonstrate targeted gene insertion of marker-free DNA in rice using CRISPR-Cas9 genome editing, and offer a promising strategy for genetic improvement of rice and other crops.


Assuntos
Carotenoides/metabolismo , Edição de Genes/métodos , Técnicas de Introdução de Genes/métodos , Oryza/genética , Melhoramento Vegetal/métodos , Vias Biossintéticas/genética , Sistemas CRISPR-Cas/genética , Carotenoides/análise , DNA de Plantas/genética , Genoma de Planta/genética , Oryza/metabolismo , Plantas Geneticamente Modificadas , Sementes/química , Sequenciamento Completo do Genoma
3.
Cell ; 180(6): 1262-1271.e15, 2020 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-32169219

RESUMO

Establishing causal links between non-coding variants and human phenotypes is an increasing challenge. Here, we introduce a high-throughput mouse reporter assay for assessing the pathogenic potential of human enhancer variants in vivo and examine nearly a thousand variants in an enhancer repeatedly linked to polydactyly. We show that 71% of all rare non-coding variants previously proposed as causal lead to reporter gene expression in a pattern consistent with their pathogenic role. Variants observed to alter enhancer activity were further confirmed to cause polydactyly in knockin mice. We also used combinatorial and single-nucleotide mutagenesis to evaluate the in vivo impact of mutations affecting all positions of the enhancer and identified additional functional substitutions, including potentially pathogenic variants hitherto not observed in humans. Our results uncover the functional consequences of hundreds of mutations in a phenotype-associated enhancer and establish a widely applicable strategy for systematic in vivo evaluation of human enhancer variants.


Assuntos
Elementos Facilitadores Genéticos/genética , Ensaios de Triagem em Larga Escala/métodos , Polidactilia/genética , Animais , Elementos Facilitadores Genéticos/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Técnicas de Introdução de Genes/métodos , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Humanos , Camundongos , Mutação , Fenótipo , Polidactilia/metabolismo , RNA não Traduzido/genética
4.
Biochem J ; 477(5): 887-903, 2020 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-32003433

RESUMO

So far, it has been still unknown how liamocins are biosynthesized, regulated, transported and secreted. In this study, a highly reducing polyketide synthase (HR-PKS), a mannitol-1-phosphate dehydrogenase (MPDH), a mannitol dehydrogenase (MtDH), an arabitol dehydrogenase (ArDH) and an esterase (Est1) were found to be closely related to core biosynthesis of extracellular liamocins in Aureobasidium melanogenum 6-1-2. The HR-PKS was responsible for biosynthesis of 3,5-dihydroxydecanoic acid. The MPDH and MtDH were implicated in mannitol biosynthesis and the ArDH was involved in arabitol biosynthesis. The Est1 catalyzed ester bond formation of them. A phosphopantetheine transferase (PPTase) activated the HR-PKS and a transcriptional activator Ga11 activated expression of the PKS1 gene. Therefore, deletion of the PKS1 gene, all the three genes encoding MPDH, MtDH and ArDH, the EST1, the gene responsible for PPTase and the gene for Ga11 made all the disruptants (Δpks13, Δpta13, Δest1, Δp12 and Δg11) totally lose the ability to produce any liamocins. A GLTP gene encoding a glycolipid transporter and a MDR1 gene encoding an ABC transporter took part in transport and secretion of the produced liamocins into medium. Removal of the GLTP gene and the MDR1 gene resulted in a Δgltp1 mutant and a Δmdr16 mutant, respectively, that lost the partial ability to secrete liamocins, but which cells were swollen and intracellular lipid accumulation was greatly enhanced. Hydrolysis of liamocins released 3,5-dihydroxydecanoic acid, mannitol, arabitol and acetic acid. We proposed a core biosynthesis pathway, regulation, transport and secretion of liamocins in A. melanogenum.


Assuntos
Ascomicetos/genética , Ascomicetos/metabolismo , Vias Biossintéticas/fisiologia , Manitol/análogos & derivados , Óleos/metabolismo , Transporte Proteico/fisiologia , Técnicas de Introdução de Genes/métodos , Manitol/análise , Manitol/metabolismo , Óleos/análise
5.
Scand J Immunol ; 91(4): e12862, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31889332

RESUMO

CRISPR/Cas9 is a powerful gene-editing tool allowing for specific gene manipulation at targeted sites in the genome. Here, we used CRISPR/Cas9-mediated gene editing to introduce single amino acid mutations into proteins involved in T cell receptor signalling pathways. Knock-in mutations were introduced in Jurkat T cells by homologous directed repair using single-stranded oligodeoxynucleotides. Specifically, we aimed to create targeted mutations at two loci within LCK, a constitutively expressed gene, and at three loci within SH2D2A, whose expression is induced upon T cell activation. Here, we present a simple workflow that can be applied by any laboratory equipped for cell culture work, utilizing basic flow cytometry, Western blotting and PCR techniques. Our data reveal that gene editing may be locus-dependent and can vary between target sites, also within a gene. In our two targeted genes, on average 2% of the clones harboured homozygous mutations as assessed by allele-specific PCR and subsequent sequencing. We highlight the importance of decreasing the clonal heterogeneity and developing robust screening methods to accurately select for correct knock-in mutations. Our workflow may be employed in other immune cell lines and acts as a useful approach for decoding functional mechanisms of proteins of interest.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes/métodos , Técnicas de Introdução de Genes/métodos , Linfócitos T , Fluxo de Trabalho , Humanos , Células Jurkat , Mutação
6.
Curr Protoc Mouse Biol ; 10(1): e67, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31912993

RESUMO

Generating large-fragment knock-ins, such as reporters, conditional alleles, or humanized alleles, directly in mouse embryos is still a challenging feat. We have developed 2C-HR-CRISPR, a technology that allows highly efficient (10-50%) and rapid (generating founders in 2 months) targeting of large DNA fragments. Key to this strategy is the delivery of CRISPR reagents into 2-cell-stage mouse embryos, taking advantage of the high homologous recombination activity during the long G2 cell cycle phase at this stage. Furthermore, by exploiting a Cas9-monomeric streptavidin (Cas-mSA) and biotinylated PCR template (BioPCR) system to localize the repair template to specific double strand breaks, the efficiency can be further improved to up to 95%. Here we provide a procedure to generate large-fragment knock-in mouse models using 2C-HR-CRISPR. We first describe the principles for designing single guide RNAs and repair templates but refer to published manuscripts and protocols for molecular cloning methods or commercial sources for these reagents. We then describe two unique aspects of 2C-HR-CRISPR that are critical for success: (1) production of the CRISPR reagents for 2C-HR-CRISPR, particularly for applying the Cas9-mSA/BioPCR method, and (2) microinjection of mouse embryos at the 2-cell stage. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Single guide RNA and repair template design Basic Protocol 2: Preparing reagents for 2C-HR-CRISPR Basic Protocol 3: Microinjecting 2-cell-stage mouse embryos.


Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Técnicas de Introdução de Genes/métodos , Recombinação Homóloga , Camundongos Transgênicos/genética , Modelos Animais , Animais , Camundongos
7.
Cell Rep ; 30(4): 1195-1207.e7, 2020 01 28.
Artigo em Inglês | MEDLINE | ID: mdl-31995758

RESUMO

Targeted knockin mediated by double-stranded DNA cleavage is accompanied by unwanted insertions and deletions (indels) at on-target and off-target sites. A nick-mediated approach scarcely generates indels but exhibits reduced efficiency of targeted knockin. Here, we demonstrate that tandem paired nicking, a method for targeted knockin involving two Cas9 nickases that create nicks at the homologous regions of the donor DNA and the genome in the same strand, scarcely creates indels at the edited genomic loci, while permitting the efficiency of targeted knockin largely equivalent to that of the Cas9-nuclease-based approach. Tandem paired nicking seems to accomplish targeted knockin by DNA recombination analogous to Holliday's model and creates intended genomic changes without introducing additional nucleotide changes, such as silent mutations. Targeted knockin through tandem paired nicking neither triggers significant p53 activation nor occurs preferentially in p53-suppressed cells. These properties of tandem paired nicking demonstrate its utility in precision genome engineering.


Assuntos
Caspase 9/metabolismo , Desoxirribonuclease I/metabolismo , Edição de Genes/métodos , Marcação de Genes/métodos , Proteína Supressora de Tumor p53/metabolismo , Quebras de DNA de Cadeia Dupla , DNA Cruciforme , Técnicas de Introdução de Genes/métodos , Células HEK293 , Células HeLa , Humanos , Mutação INDEL , Recombinação Genética , Proteína Supressora de Tumor p53/genética
8.
Methods Mol Biol ; 2041: 17-43, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31646478

RESUMO

Purinergic signaling involves extracellular purines and pyrimidines acting upon specific cell surface purinoceptors classified into the P1, P2X, and P2Y families for nucleosides and nucleotides. This widespread signaling mechanism is active in all major tissues and influences a range of functions in health and disease. Orthologs to all but one of the human purinoceptors have been found in mouse, making this laboratory animal a useful model to study their function. Indeed, analyses of purinoceptors via knock-in or knockout approaches to produce gain or loss of function phenotypes have revealed several important therapeutic targets. None of the homozygous purinoceptor knockouts proved to be developmentally lethal, which suggest that either these receptors are not involved in key developmental processes or that the large number of receptors in each family allowed for functional compensation. Different models for the same purinoceptor often show compatible phenotypes but there have been examples of significant discrepancies. These revealed unexpected differences in the structure of human and mouse genes and emphasized the importance of the genetic background of different mouse strains. In this chapter, we provide an overview of the current knowledge and new trends in the modifications of purinoceptor genes in vivo. We discuss the resulting phenotypes, their applications and relative merits and limitations of mouse models available to study purinoceptor subtypes.


Assuntos
Técnicas de Introdução de Genes/métodos , Modelos Animais , Purinas/metabolismo , Receptores Purinérgicos/metabolismo , Transdução de Sinais , Trifosfato de Adenosina/metabolismo , Animais , Humanos , Camundongos , Camundongos Knockout
9.
Sci China Life Sci ; 63(1): 59-67, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31872378

RESUMO

The zebrafish has become a popular vertebrate animal model in biomedical research. However, it is still challenging to make conditional gene knockout (CKO) models in zebrafish due to the low efficiency of homologous recombination (HR). Here we report an efficient non-HR-based method for generating zebrafish carrying a CKO and knockin (KI) switch (zCKOIS) coupled with dual-color fluorescent reporters. Using this strategy, we generated hey2zKOIS which served as a hey2 KI reporter with EGFP expression. Upon Cre induction in targeted cells, the hey2zCKOIS was switched to a non-functional CKO allele hey2zCKOIS-invassociated with TagRFP expression, enabling visualization of the CKO alleles. Thus, simplification of the design, and the visibility and combination of both CKO and KI alleles make our zCKOIS strategy an applicable CKO approach for zebrafish.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Sistemas CRISPR-Cas/genética , Marcação de Genes/métodos , Recombinação Homóloga/genética , Íntrons/genética , Proteínas de Peixe-Zebra/genética , Alelos , Animais , Animais Geneticamente Modificados , Sequência de Bases , Expressão Gênica , Técnicas de Introdução de Genes/métodos , Técnicas de Inativação de Genes/métodos , Engenharia Genética , Genótipo , Proteínas de Fluorescência Verde/genética , Peixe-Zebra
10.
Nat Chem Biol ; 16(4): 387-390, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31873222

RESUMO

Here, we report a rapid CRISPR-Cas9-mediated gene knock-in strategy that uses Cas9 ribonucleoprotein and 5'-modified double-stranded DNA donors with 50-base-pair homology arms and achieved unprecedented 65/40% knock-in rates for 0.7/2.5 kilobase inserts, respectively, in human embryonic kidney 293T cells. The identified 5'-end modification led to up to a fivefold increase in gene knock-in rates at various genomic loci in human cancer and stem cells.


Assuntos
Técnicas de Introdução de Genes/métodos , Região 5'-Flanqueadora/genética , Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , DNA/genética , Genoma/genética , Células HEK293 , Humanos , RNA Guia/genética , Homologia de Sequência do Ácido Nucleico
11.
Cells ; 8(12)2019 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-31847480

RESUMO

Cell plasticity, defined as the ability to undergo phenotypical transformation in a reversible manner, is a physiological process that also exerts important roles in disease progression. Two forms of cellular plasticity are epithelial-mesenchymal transition (EMT) and its inverse process, mesenchymal-epithelial transition (MET). These processes have been correlated to the poor outcome of different types of neoplasias as well as drug resistance development. Since EMT/MET are transitional processes, we generated and validated a reporter cell line. Specifically, a far-red fluorescent protein was knocked-in in-frame with the mesenchymal gene marker VIMENTIN (VIM) in H2170 lung cancer cells. The vimentin reporter cells (VRCs) are a reliable model for studying EMT and MET showing cellular plasticity upon a series of stimulations. These cells are a robust platform to dissect the molecular mechanisms of these processes, and for drug discovery in vitro and in vivo in the future.


Assuntos
Transição Epitelial-Mesenquimal/genética , Engenharia Genética/métodos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Caderinas/metabolismo , Linhagem Celular Tumoral , Plasticidade Celular/genética , Técnicas de Introdução de Genes/métodos , Células HEK293 , Humanos , Proteínas Luminescentes/genética , Vimentina/genética , Vimentina/metabolismo
12.
Elife ; 82019 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-31674908

RESUMO

We previously reported a CRISPR-mediated knock-in strategy into introns of Drosophila genes, generating an attP-FRT-SA-T2A-GAL4-polyA-3XP3-EGFP-FRT-attP transgenic library for multiple uses (Lee et al., 2018a). The method relied on double stranded DNA (dsDNA) homology donors with ~1 kb homology arms. Here, we describe three new simpler ways to edit genes in flies. We create single stranded DNA (ssDNA) donors using PCR and add 100 nt of homology on each side of an integration cassette, followed by enzymatic removal of one strand. Using this method, we generated GFP-tagged proteins that mark organelles in S2 cells. We then describe two dsDNA methods using cheap synthesized donors flanked by 100 nt homology arms and gRNA target sites cloned into a plasmid. Upon injection, donor DNA (1 to 5 kb) is released from the plasmid by Cas9. The cassette integrates efficiently and precisely in vivo. The approach is fast, cheap, and scalable.


Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Técnicas de Introdução de Genes/métodos , Recombinação Homóloga , Mutagênese Insercional/métodos , Animais , DNA/genética , DNA de Cadeia Simples/genética , Drosophila
13.
J Neuroinflammation ; 16(1): 173, 2019 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-31470863

RESUMO

BACKGROUND: Disturbances in clock genes affect almost all patients with Alzheimer's disease (AD), as evidenced by their altered sleep/wake cycle, thermoregulation, and exacerbation of cognitive impairment. As microglia-mediated neuroinflammation proved to be a driver of AD rather than a result of the disease, in this study, we evaluated the relationship between clock gene disturbance and neuroinflammation in microglia and their contribution to the onset of AD. METHODS: In this study, the expression of clock genes and inflammatory-related genes was examined in MACS microglia isolated from 2-month-old amyloid precursor protein knock-in (APP-KI) and wild-type (WT) mice using cap analysis gene expression (CAGE) deep sequencing and RT-PCR. The effects of clock gene disturbance on neuroinflammation and relevant memory changes were examined in 2-month-old APP-KI and WT mice after injection with SR9009 (a synthetic agonist for REV-ERB). The microglia morphology was studied by staining, neuroinflammation was examined by Western blotting, and cognitive changes were examined by Y-maze and novel object recognition tests. RESULTS: CLOCK/BMAL1-driven transcriptional negative feedback loops were impaired in the microglia from 2-month-old APP-KI mice. Pro-inflammatory genes in microglia isolated from APP-KI mice were significantly higher than those isolated from WT mice at Zeitgeber time 14. The expression of pro-inflammatory genes was positively associated with NF-κB activation and negatively associated with the BMAL1 expression. SR9009 induced the activation of microglia, the increased expression of pro-inflammatory genes, and cognitive decline in 2-month-old APP-KI mice. CONCLUSION: Clock gene disturbance in microglia is involved in the early onset of AD through the induction of chronic neuroinflammation, which may be a new target for preventing or slowing AD.


Assuntos
Precursor de Proteína beta-Amiloide/metabolismo , Encéfalo/metabolismo , Proteínas CLOCK/metabolismo , Técnicas de Introdução de Genes/métodos , Mediadores da Inflamação/metabolismo , Microglia/metabolismo , Precursor de Proteína beta-Amiloide/genética , Animais , Encéfalo/efeitos dos fármacos , Proteínas CLOCK/antagonistas & inibidores , Proteínas CLOCK/genética , Inflamação/genética , Inflamação/metabolismo , Mediadores da Inflamação/agonistas , Locomoção/efeitos dos fármacos , Locomoção/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microglia/efeitos dos fármacos , Pirrolidinas/toxicidade , Tiofenos/toxicidade
14.
Genes (Basel) ; 10(9)2019 08 29.
Artigo em Inglês | MEDLINE | ID: mdl-31470649

RESUMO

The ectopic overexpression of foreign genes in animal genomes is an important strategy for gain-of-function study and establishment of transgenic animal models. Previous studies showed that two loci (Rosa26 and pH11) were identified as safe harbor locus in pig genomes, which means foreign genes can be integrated into this locus for stable expression. Moreover, integration of a transgene may interfere with the endogenous gene expression of the target locus after the foreign fragments are inserted. Here, we provide a new strategy for efficient transgene knock-in in the endogenous GAPDH gene via CRISPR/Cas9 mediated homologous recombination. This strategy has no influence on the expression of the endogenous GAPDH gene. Thus, the GAPDH locus is a new alternative safe harbor locus in the pig genome for foreign gene knock-ins. This strategy is promising for agricultural breeding and biomedical model applications.


Assuntos
Técnicas de Introdução de Genes/métodos , Gliceraldeído-3-Fosfato Desidrogenases/genética , Suínos/genética , Animais , Sistemas CRISPR-Cas , Técnicas de Introdução de Genes/efeitos adversos , Genoma
15.
eNeuro ; 6(5)2019.
Artigo em Inglês | MEDLINE | ID: mdl-31451604

RESUMO

Genetically modified mice have become standard tools in neuroscience research. Our understanding of the basal ganglia in particular has been greatly assisted by BAC mutants with selective transgene expression in striatal neurons forming the direct or indirect pathways. However, for more sophisticated behavioral tasks and larger intracranial implants, rat models are preferred. Furthermore, BAC lines can show variable expression patterns depending upon genomic insertion site. We therefore used CRISPR/Cas9 to generate two novel knock-in rat lines specifically encoding Cre recombinase immediately after the dopamine D1 receptor (Drd1a) or adenosine 2a receptor (Adora2a) loci. Here, we validate these lines using in situ hybridization and viral vector mediated transfection to demonstrate selective, functional Cre expression in the striatal direct and indirect pathways, respectively. We used whole-genome sequencing to confirm the lack of off-target effects and established that both rat lines have normal locomotor activity and learning in simple instrumental and Pavlovian tasks. We expect these new D1-Cre and A2a-Cre rat lines will be widely used to study both normal brain functions and neurological and psychiatric pathophysiology.


Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Integrases/genética , Receptor A2A de Adenosina/genética , Receptores de Dopamina D1/genética , Animais , Feminino , Técnicas de Introdução de Genes/métodos , Integrases/biossíntese , Masculino , Ratos , Ratos Long-Evans , Ratos Transgênicos , Receptor A2A de Adenosina/biossíntese , Receptores de Dopamina D1/biossíntese
16.
PLoS One ; 14(8): e0221164, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31454364

RESUMO

Knock-in (KI) gene targeting can be employed for a wide range of applications in stem cell research. However, vectors for KI require multiple complicated processes for construction, including multiple times of digestion/ligation steps and extensive restriction mapping, which has imposed limitations for the robust applicability of KI gene targeting. To circumvent this issue, here we introduce versatile and systematic methods for generating KI vectors by molecular cloning. In this approach, we employed the Multisite Gateway technology, an efficient in vitro DNA recombination system using proprietary sequences and enzymes. KI vector construction exploiting these methods requires only efficient steps, such as PCR and recombination, enabling robust KI gene targeting. We show that combinatorial usage of the KI vectors generated using this method and site-specific nucleases enabled the precise integration of fluorescent protein genes in multiple loci of human and common marmoset (marmoset; Callithrix jacchus) pluripotent stem cells. The methods described here will facilitate the usage of KI technology and ultimately help to accelerate stem cell research.


Assuntos
DNA Recombinante/genética , Técnicas de Introdução de Genes/métodos , Marcação de Genes/métodos , Vetores Genéticos/genética , Animais , Callithrix/genética , Clonagem Molecular/métodos , Desoxirribonucleases/genética , Genes Reporter/genética , Proteínas de Fluorescência Verde/genética , Humanos , Pesquisa com Células-Tronco
17.
eNeuro ; 6(4)2019.
Artigo em Inglês | MEDLINE | ID: mdl-31371457

RESUMO

Microglia are specialized brain-resident macrophages with important functions in health and disease. To improve our understanding of these cells, the research community needs genetic tools to identify and control them in a manner that distinguishes them from closely related cell types. We have targeted the recently discovered microglia-specific Tmem119 gene to generate knock-in mice expressing EGFP (JAX#031823) or CreERT2 (JAX#031820) for the identification and manipulation of microglia, respectively. Genetic characterization of the locus and qPCR-based analysis demonstrate correct positioning of the transgenes and intact expression of endogenous Tmem119 in the knock-in mouse models. Immunofluorescence analysis further shows that parenchymal microglia, but not other brain macrophages, are completely and faithfully labeled in the EGFP-line at different time points of development. Flow cytometry indicates highly selective expression of EGFP in CD11b+CD45lo microglia. Similarly, immunofluorescence and flow cytometry analyses using a Cre-dependent reporter mouse line demonstrate activity of CreERT2 primarily in microglia upon tamoxifen administration with the caveat of activity in leptomeningeal cells. Finally, flow cytometric analyses reveal absence of EGFP expression and minimal activity of CreERT2 in blood monocytes of the Tmem119-EGFP and Tmem119-CreERT2 lines, respectively. These new transgenic lines extend the microglia toolbox by providing the currently most specific genetic labeling and control over these cells in the myeloid compartment of mice.


Assuntos
Técnicas de Introdução de Genes/métodos , Proteínas de Membrana/genética , Microglia/metabolismo , Animais , Feminino , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Imuno-Histoquímica/métodos , Integrases/genética , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microglia/efeitos dos fármacos , Monócitos/metabolismo , Tamoxifeno/administração & dosagem
18.
J Biol Chem ; 294(37): 13718-13728, 2019 09 13.
Artigo em Inglês | MEDLINE | ID: mdl-31346037

RESUMO

Protein methyltransferases mediate posttranslational modifications of both histone and nonhistone proteins. Whereas histone methylation is well-known to regulate gene expression, the biological significance of nonhistone methylation is poorly understood. Methyltransferase-like 21c (Mettl21c) is a newly classified nonhistone lysine methyltransferase whose in vivo function has remained elusive. Using a Mettl21c LacZ knockin mouse model, we show here that Mettl21c expression is absent during myogenesis and restricted to mature type I (slow) myofibers in the muscle. Using co-immunoprecipitation, MS, and methylation assays, we demonstrate that Mettl21c trimethylates heat shock protein 8 (Hspa8) at Lys-561 to enhance its stability. As such, Mettl21c knockout reduced Hspa8 trimethylation and protein levels in slow muscles, and Mettl21c overexpression in myoblasts increased Hspa8 trimethylation and protein levels. We further show that Mettl21c-mediated stabilization of Hspa8 enhances its function in chaperone-mediated autophagy, leading to degradation of client proteins such as the transcription factors myocyte enhancer factor 2A (Mef2A) and Mef2D. In contrast, Mettl21c knockout increased Mef2 protein levels in slow muscles. These results identify Hspa8 as a Mettl21c substrate and reveal that nonhistone methylation has a physiological function in protein stabilization.


Assuntos
Proteínas de Choque Térmico HSC70/metabolismo , Metiltransferases/metabolismo , Miofibrilas/metabolismo , Animais , Autofagia , Feminino , Técnicas de Introdução de Genes/métodos , Células HEK293 , Proteínas de Choque Térmico HSC70/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Fatores de Transcrição MEF2/genética , Masculino , Metilação , Metiltransferases/genética , Camundongos , Desenvolvimento Muscular/genética , Músculos/metabolismo , Mioblastos/metabolismo , Miofibrilas/genética , Processamento de Proteína Pós-Traducional
20.
Purinergic Signal ; 15(3): 397-402, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31286385

RESUMO

A P2X1-eYFP knock-in mouse was generated to study receptor expression and mobility in smooth muscle and blood cells. eYFP was added to the C-terminus of the P2X1R and replaced the native P2X1R. Fluorescence corresponding to P2X1-eYFPR was detected in urinary bladder smooth muscle, platelets and megakaryocytes. ATP-evoked currents from wild type and P2X1-eYFP isolated urinary bladder smooth muscle cells had the same peak current amplitude and time-course showing that the eYFP addition had no obvious effect on properties. Fluorescence recovery after photobleaching (FRAP) in bladder smooth muscle cells demonstrated that surface P2X1Rs are mobile and their movement is reduced following cholesterol depletion. Compared to the platelet and megakaryocyte, P2X1-eYFP fluorescence was negligible in red blood cells and the majority of smaller marrow cells. The spatial pattern of P2X1-eYFP fluorescence in the megakaryocyte along with FRAP assessment of mobility suggested that P2X1Rs are expressed extensively throughout the membrane invagination system of this cell type. The current study highlights that the spatiotemporal properties of P2X1R expression can be monitored in real time in smooth muscle cells and megakaryocytes/platelets using the eYFP knock-in mouse model.


Assuntos
Técnicas de Introdução de Genes/métodos , Receptores Purinérgicos P2X1/análise , Receptores Purinérgicos P2X1/metabolismo , Animais , Proteínas de Bactérias , Proteínas Luminescentes , Camundongos , Modelos Animais
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