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1.
Int J Mol Sci ; 22(15)2021 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-34360910

RESUMO

Modified mRNA (modRNA)-based somatic reprogramming is an effective and safe approach that overcomes the genomic mutation risk caused by viral integrative methods. It has improved the disadvantages of conventional mRNA and has better stability and immunogenicity. The modRNA molecules encoding multiple pluripotent factors have been applied successfully in reprogramming somatic cells such as fibroblasts, mesenchymal stem cells, and amniotic fluid stem cells to generate pluripotent stem cells (iPSCs). Moreover, it also can be directly used in the terminal differentiation of stem cells and fibroblasts into functional therapeutic cells, which exhibit great promise in disease modeling, drug screening, cell transplantation therapy, and regenerative medicine. In this review, we summarized the reprogramming applications of modified mRNA in iPSC generation and therapeutic applications of functionally differentiated cells.


Assuntos
Diferenciação Celular/genética , Reprogramação Celular/genética , Células-Tronco Pluripotentes Induzidas/citologia , RNA Mensageiro/genética , Transfecção/métodos , Animais , Transdiferenciação Celular/genética , Transplante de Células/métodos , Humanos , Camundongos , Medicina Regenerativa/métodos
2.
Molecules ; 26(15)2021 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-34361779

RESUMO

Delivering nucleic acids into the endothelium has great potential in treating vascular diseases. However, endothelial cells, which line the vasculature, are considered as sensitive in nature and hard to transfect. Low transfection efficacies in endothelial cells limit their potential therapeutic applications. Towards improving the transfection efficiency, we made an effort to understand the internalization of lipoplexes into the cells, which is the first and most critical step in nucleic acid transfections. In this study, we demonstrated that the transient modulation of caveolae/lipid rafts mediated endocytosis with the cholesterol-sequestrating agents, nystatin, filipin III, and siRNA against Cav-1, which significantly increased the transfection properties of cationic lipid-(2-hydroxy-N-methyl-N,N-bis(2-tetradecanamidoethyl)ethanaminium chloride), namely, amide liposomes in combination with 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) (AD Liposomes) in liver sinusoidal endothelial cells (SK-Hep1). In particular, nystatin was found to be highly effective with 2-3-fold enhanced transfection efficacy when compared with amide liposomes in combination with Cholesterol (AC), by switching lipoplex internalization predominantly through clathrin-mediated endocytosis and macropinocytosis.


Assuntos
Cavéolas/efeitos dos fármacos , Colesterol/química , Células Endoteliais/efeitos dos fármacos , Lipossomos/química , Microdomínios da Membrana/efeitos dos fármacos , Transfecção/métodos , Animais , Cavéolas/química , Cavéolas/metabolismo , Caveolina 1/antagonistas & inibidores , Caveolina 1/genética , Caveolina 1/metabolismo , Linhagem Celular Transformada , Colesterol/metabolismo , Clatrina/metabolismo , DNA/química , DNA/metabolismo , Endocitose/efeitos dos fármacos , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Filipina/química , Filipina/farmacologia , Expressão Gênica , Lipossomos/metabolismo , Microdomínios da Membrana/química , Microdomínios da Membrana/metabolismo , Nistatina/química , Nistatina/farmacologia , Fosfatidiletanolaminas/química , Fosfatidiletanolaminas/farmacologia , Pinocitose/efeitos dos fármacos , Plasmídeos/química , Plasmídeos/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ratos
3.
Anticancer Res ; 41(8): 3731-3740, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34281831

RESUMO

BACKGROUND: The clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR-Cas9) is thought to have promising clinical potential. However, the off-target effects of Cas9 are a major concern for its application. Therefore, we hypothesized that the adverse effects of off-target gene editing might be minimized if the human codon-optimized Streptococcus pyogenes Cas9 (hCas9) could be specifically expressed in cancer cells. MATERIALS AND METHODS: We constructed a chimeric adenoviral vector, Ad5F35-MKp-hCas9, and infected human bladder cancer cell lines with this vector. The confirmation of hCas9 gene expression was performed in 3-4 days after from infection. RESULTS: hCas9 gene expression was observed in Ad5F35-MKp-hCas9 infected bladder cancer cells but not in non-malignant cells. CONCLUSION: Our study showed that the Ad5F35-MKp-hCas9 vector is capable of expressing the hCas9 gene with high specificity in bladder cancer cells. These findings may help in minimizing the risk of off-target effects of gene editing.


Assuntos
Adenoviridae/genética , Proteína 9 Associada à CRISPR/genética , Vetores Genéticos/genética , Transfecção/métodos , Neoplasias da Bexiga Urinária/genética , Proteína 9 Associada à CRISPR/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Edição de Genes/métodos , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Humanos , Regiões Promotoras Genéticas , Neoplasias da Bexiga Urinária/patologia
4.
Methods Mol Biol ; 2277: 49-67, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34080144

RESUMO

Defects in human mitochondrial genome can cause a wide range of clinical disorders that still do not have efficient therapies. The natural pathway of small noncoding RNA import can be exploited to address therapeutic RNAs into the mitochondria. To create an approach of carrier-free targeting of RNA into living human cells, we designed conjugates containing a cholesterol residue and developed the protocols of chemical synthesis of oligoribonucleotides conjugated with cholesterol residue through cleavable pH-triggered hydrazone bond. The biodegradable conjugates of importable RNA with cholesterol can be internalized by cells in a carrier-free manner; RNA can then be released in the late endosomes due to a change in pH and partially targeted into mitochondria. Here we provide detailed protocols for solid-phase and "in solution" chemical synthesis of oligoribonucleotides conjugated to a cholesterol residue through a hydrazone bond. We describe the optimization of the carrier-free cell transfection with these conjugated RNA molecules and methods for evaluating the cellular and mitochondrial uptake of lipophilic conjugates.


Assuntos
Mitocôndrias/genética , Oligorribonucleotídeos/síntese química , RNA/química , Transfecção/métodos , Células Cultivadas , Colesterol/química , Humanos , Hidrazonas/química , Concentração de Íons de Hidrogênio , RNA/administração & dosagem
5.
Bioelectrochemistry ; 140: 107803, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-33975183

RESUMO

High-frequency bipolar pulses (HF-BP) have been demonstrated to be efficient for membrane permeabilization and irreversible electroporation. Since membrane permeabilization has been achieved using HF-BP pulses we hypothesized that with these pulses we can also achieve successful gene electrotransfer (GET). Three variations of bursts of 2 µs bipolar pulses with 2 µs interphase delay were applied in HF-BP protocols. We compared transfection efficiency of monopolar micro and millisecond pulses and HF-BP protocols at various plasmid DNA (pDNA) concentrations on CHO - K1 cells. GET efficiency increased with increasing pDNA concentration. Overall GET obtained by HF-BP pulse protocols was comparable to overall GET obtained by longer monopolar pulse protocols. Our results, however, suggest that although we were able to achieve similar percent of transfected cells, the number of pDNA copies that were successfully transferred into cells seemed to be higher when longer monopolar pulses were used. Interestingly, we did not observe any direct correlation between fluorescence intensity of pDNA aggregates formed on cell membrane and transfection efficiency. The results of our study confirmed that we can achieve successful GET with bipolar microsecond i. e. HF-BP pulses, although at the expense of higher pDNA concentrations.


Assuntos
Eletroporação/métodos , Transfecção/métodos , DNA/genética , Plasmídeos/genética , Espectrometria de Fluorescência
6.
Methods Mol Biol ; 2305: 83-104, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33950385

RESUMO

Mammalian protein expression systems are ideally suited for the high-level production of recombinant eukaryotic secreted and membrane proteins for structural biology applications. Here, we present genetic transduction of HEK293-derived cells using lentivirus as a robust and cost-efficient method for the rapid generation of stable expression cell lines. We describe the features of the lentiviral transfer plasmid pHR-CMV-TetO2, as well as detailed protocols for production of lentiviral particles, determination of functional lentiviral titer, infection of expression cells, culture and expansion of the resulting stable cell lines, their adaptation to adherent and suspension growth, and constitutive or inducible milligram-scale protein production. The typical lead-time for a full production run is ~3-4 weeks, with an anticipated yield of up to tens of milligrams of protein per liter of expression medium.


Assuntos
Biotecnologia/métodos , Técnicas de Cultura de Células/métodos , Lentivirus/metabolismo , Proteínas Recombinantes/biossíntese , Animais , Linhagem Celular , Vetores Genéticos , Células HEK293 , Humanos , Lentivirus/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Plasmídeos/genética , Proteínas Recombinantes/genética , Transdução Genética/métodos , Transfecção/métodos
7.
Methods Mol Biol ; 2305: 105-128, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33950386

RESUMO

Cancers, neurodegenerative and infectious diseases remain some of the leading causes of deaths worldwide. The structure-guided drug design is essential to advance drug development for these important diseases. One of the key challenges in the structure determination workflow is the production of eukaryotic membrane proteins (drug targets) of high quality. A number of expression systems have been developed for the production of eukaryotic membrane proteins. In this chapter, an optimized detailed protocol for transient transfection and expression of eukaryotic membrane proteins in Expi293F cells is presented. Testing expression and purification on a small scale allow optimizing conditions for sample preparation for downstream structural (cryo-EM) elucidation.


Assuntos
Biotecnologia/métodos , Técnicas de Cultura de Células/métodos , Células Eucarióticas/metabolismo , Proteínas de Membrana/biossíntese , Proteínas Recombinantes de Fusão/biossíntese , Linhagem Celular , Cromatografia em Gel , Eucariotos/genética , Eucariotos/metabolismo , Expressão Gênica , Células HEK293 , Humanos , Proteínas de Membrana/genética , Proteínas Recombinantes de Fusão/genética , Transfecção/métodos
8.
Methods Mol Biol ; 2305: 129-140, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33950387

RESUMO

The expression of mammalian recombinant proteins in insect cell lines using transient-plasmid-based gene expression enables the production of high-quality protein samples. Here, the procedure for virus-free transient gene expression (TGE) in High Five insect cells is described in detail. The parameters that determine the efficiency and reproducibility of the method are presented in a robust protocol for easy implementation and set-up of the method. The applicability of the TGE method in High Five cells for proteomic, structural, and functional analysis of the expressed proteins is shown.


Assuntos
Biotecnologia/métodos , Clonagem Molecular , Insetos/metabolismo , Glicoproteína da Espícula de Coronavírus/biossíntese , Transfecção/métodos , Animais , Reatores Biológicos , Técnicas de Cultura de Células/métodos , Linhagem Celular , Expressão Gênica , Glicosilação , Humanos , Insetos/citologia , Mamíferos/genética , Mamíferos/metabolismo , Plasmídeos , Proteômica , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Reprodutibilidade dos Testes , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/genética
9.
BMC Cancer ; 21(1): 483, 2021 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-33931030

RESUMO

BACKGROUND: Nasopharyngeal carcinoma (NPC) is a malignant head and neck tumor, and more than 70% of new cases are in East and Southeast Asia. However, association between NPC and pseudogenes playing important roles in genesis of multiple tumor types is still not clear and needs to be investigated. METHODS: Using RNA-Sequencing (RNA-seq) technology, we analyzed pseudogene expression in 13 primary NPC and 6 recurrent NPC samples as well as their paracancerous counterparts. Quantitative PCR was used to validate the differentially expressed pseudogenes. RESULTS: We found 251 differentially expressed pseudogenes including 73 up-regulated and 178 down-regulated ones between primary NPC and paracancerous tissues. Enrichment analysis of gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway were conducted to filter out the key pseudogenes. We reported that pseudogenes from cytochrome P450 (CYP) family, such as CYP2F2P, CYP2G1P, CYP4F24P, CYP2B7P and CYP2G2P were significantly down-regulated in NPC compared to paracancerous tissues, while IGHV1OR15-2, IGHV3-11, FCGR1CP and IGHV3-69-1 belonging to Fc gamma receptors were significantly up-regulated. CYP2B7P, CYP2F2P and CYP4F26P were enriched in arachidonic acid metabolism pathway. The qRT-PCR analysis validated the lower expression of pseudogenes CYP2F2P and CYP2B7P in NPC tissues and cell lines compared to paracancerous tissues and normal human nasopharyngeal epithelial cell line. CYP2B7P overexpression weakened migratory and invasive capacity of NPC cell line. Moreover, the expression pattern of those pseudogenes in recurrent NPC tissues was different from the primary NPC. CONCLUSION: This study suggested the role of pseudogenes in tumorigenesis and progression, potentially functioning as therapeutic targets to NPC.


Assuntos
Sistema Enzimático do Citocromo P-450/genética , Carcinoma Nasofaríngeo/genética , Neoplasias Nasofaríngeas/genética , Recidiva Local de Neoplasia/genética , Pseudogenes , Receptores de IgG/genética , Análise de Sequência de RNA , Adulto , Idoso , Ácido Araquidônico/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Família 2 do Citocromo P450/genética , Regulação para Baixo , Feminino , Ontologia Genética , Humanos , Masculino , Pessoa de Meia-Idade , Carcinoma Nasofaríngeo/patologia , Neoplasias Nasofaríngeas/patologia , Invasividade Neoplásica , Pseudogenes/fisiologia , Reação em Cadeia da Polimerase em Tempo Real , Transfecção/métodos , Regulação para Cima
10.
Plant Cell Rep ; 40(7): 1171-1179, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33948685

RESUMO

KEY MESSAGE: Microwounding pre-treatment facilitates agroinfiltration and transient gene expression in hard-to-agroinfiltrate citrus varieties. Agrobacterium infiltration is a widely used method for transient expression studies in plants, but this method is not used extensively in citrus because of its low efficiency. In this study, we developed an easy, cheap, and reliable agroinfiltration method for transient gene expression in citrus. A microneedle roller was used to create microscopic wounds in the leaf epidermis to facilitate agroinfiltration. Several optimization parameters were explored in this study, including the density of wounds per cm2 of abaxial leaf area, the leaf maturity grade, the effect of the Agrobacterium strain, and the length of the incubation period. Increasing the density of wounds on the leaf surface had a positive effect on transient expression. Higher transient expression levels were observed in well-expanded young leaves in comparison with older leaves. The Agrobacterium strain GV2260 was the most suitable to express a large amount of recombinant protein, and an eight- to ten-day incubation period resulted in the highest expression. Endoplasmic reticulum and cytoskeleton-targeted GFP were both successfully localized, confirming that this protocol can be used for protein subcellular localization in citrus. Finally, up to 100 ng of GFP per milligram of agroinfiltrated leaf tissue was estimated to be expressed using this method. This protocol was tested for GFP expression in five different citrus varieties with no significant statistical differences among them. This simple and easy method can speed up functional genomic studies in citrus and may be applied to other recalcitrant species with extensive epidermal cuticular wax.


Assuntos
Agrobacterium/genética , Citrus/genética , Folhas de Planta/genética , Proteínas Recombinantes/genética , DNA Bacteriano/administração & dosagem , Regulação da Expressão Gênica de Plantas , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Microscopia Confocal , Folhas de Planta/citologia , Folhas de Planta/microbiologia , Plantas Geneticamente Modificadas/genética , Proteínas Recombinantes/metabolismo , Transfecção/métodos
12.
BMC Cancer ; 21(1): 456, 2021 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-33892667

RESUMO

BACKGROUND: Endometrial cancer (UCEC) is one of the most common gynecological malignancies. We previously found that overexpression of G protein α subunit 14 (GNA14) promoted UCEC growth. Krüppel-like factor 7 (KLF7) acts as an oncogene in various cancer types, whereas the connection between GNA14 and KLF7 in UCEC is unclear. We herein explored the involvement of GNA14/KLF7 in UCEC development. METHODS: Clinical relevance of GNA14, KLF7 and HAS2 in UCEC was analyzed from TCGA and by immunohistochemical staining. Knockdown and overexpression of indicated genes were conducted by transfecting the cells with siRNAs and lentivirus, respectively. mRNA and protein expression was detected by qRT-PCR and Western blot. CCK8, colony formation, cell cycle, apoptosis, transwell and wound healing were performed to check cell biology function in vitro. Tumor growth in nude mice was conducted to check in vivo function. RNA sequencing was used to determine dys-regulated genes. RESULTS: We demonstrated that GNA14 stimulated the expression of KLF7 in UCEC cells. There was a positive correlation between GNA14 and KLF7 in normal and UCEC tissues. In vitro, KLF7 promoted cell proliferation, colony formation, cell cycle progression, and migration of UCEC cells. Apoptosis was inhibited by KLF7. Xenografted tumorigenesis of UCEC cells was suppressed by KLF7 knockdown. Furthermore, RNA sequencing results showed that KLF7 regulated the expression of a large amount of genes, among which hyaluronan synthase 2 (HAS2) was downregulated in KLF7 knockdown cells. Based on TCGA database and immunoblotting assays, KLF7 positively regulated HAS2 in UCEC cells and tissues. Lastly, knockdown of HAS2 reversed the oncogenic role of KLF7 on UCEC cell proliferation, migration, and xenografted tumor development. CONCLUSION: Taken together, we reveal that GNA14/KLF7/HAS2 signaling cascade exerts tumor promoting function during UCEC development.


Assuntos
Neoplasias do Endométrio/metabolismo , Neoplasias do Endométrio/patologia , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Hialuronan Sintases/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Animais , Sequência de Bases , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Regulação para Baixo , Neoplasias do Endométrio/genética , Feminino , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Xenoenxertos , Humanos , Hialuronan Sintases/genética , Fatores de Transcrição Kruppel-Like/genética , Lentivirus , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , RNA Mensageiro/metabolismo , RNA Interferente Pequeno , Transfecção/métodos , Regulação para Cima
13.
Int J Mol Sci ; 22(8)2021 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-33917124

RESUMO

There is an increasing interest in cationic polymers as important constituents of non-viral gene delivery vectors. In the present study, we developed a versatile synthetic route for the production of covalent polymeric conjugates consisting of water-soluble depolymerized chitosan (dCS; MW 6-9 kDa) and low molecular weight polyethylenimine (PEI; 2.5 kDa linear, 1.8 kDa branched). dCS-PEI derivatives were evaluated based on their physicochemical properties, including purity, covalent bonding, solubility in aqueous media, ability for DNA condensation, and colloidal stability of the resulting polyplexes. They were complexed with non-integrating DNA vectors coding for reporter genes by simple admixing and assessed in vitro using liver-derived HuH-7 cells for their transfection efficiency and cytotoxicity. Using a rational screening cascade, a lead compound was selected (dCS-Suc-LPEI-14) displaying the best balance of biocompatibility, cytotoxicity, and transfection efficiency. Scale-up and in vivo evaluation in wild-type mice allowed for a direct comparison with a commercially available non-viral delivery vector (in vivo-jetPEI). Hepatic expression of the reporter gene luciferase resulted in liver-specific bioluminescence, upon intrabiliary infusion of the chitosan-based polyplexes, which exceeded the signal of the in vivo jetPEI reference formulation by a factor of 10. We conclude that the novel chitosan-derivative dCS-Suc-LPEI-14 shows promise and potential as an efficient polymeric conjugate for non-viral in vivo gene therapy.


Assuntos
Quitosana/química , Técnicas de Transferência de Genes , Polietilenoimina/química , Transfecção , Animais , Linhagem Celular Tumoral , Sobrevivência Celular , Fenômenos Químicos , Técnicas de Química Sintética , Coloides/química , DNA/química , Expressão Gênica , Genes Reporter , Vetores Genéticos , Humanos , Espectroscopia de Ressonância Magnética , Camundongos , Transfecção/métodos
14.
Methods Mol Biol ; 2300: 31-37, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33792869

RESUMO

The assessment of non-coding RNAs (ncRNAs) functions highly relies on loss of function studies. However, due to their exclusive or partial nuclear localization, many small and long ncRNAs are not efficiently silenced by RNA interference. Antisense LNA GapmeRs constitute a good alternative to RNAi. They allow an effective knockdown of ncRNAs with sizes greater than 80 nucleotides, regardless of their cellular localization. This chapter focuses on the silencing of two different nuclear ncRNAs (ANRIL and SATIII RNAs) in mammalian cells using antisense LNA GapmeRs with two different transfection methods: calcium phosphate-mediated transfection and LipofectamineTM 2000.


Assuntos
Oligonucleotídeos Antissenso/farmacologia , RNA Longo não Codificante/genética , Transfecção/métodos , Fosfatos de Cálcio/química , Inativação Gênica , Células HEK293 , Células HeLa , Humanos , Lipídeos/química
15.
Int J Mol Sci ; 22(5)2021 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-33806461

RESUMO

The present study aimed to synthesize novel polycationic polymers composed of N-substituted L-2,3-diaminopropionic acid residues (DAPEGs) and investigate their cell permeability, cytotoxicity, and DNA-binding ability. The most efficient cell membrane-penetrating compounds (O2Oc-Dap(GO2)n-O2Oc-NH2, where n = 4, 6, and 8) showed dsDNA binding with a binding constant in the micromolar range (0.3, 3.4, and 0.19 µM, respectively) and were not cytotoxic to HB2 and MDA-MB-231 cells. Selected compounds used in the transfection of a GFP plasmid showed high transfection efficacy and minimal cytotoxicity. Their interaction with plasmid DNA and the increasing length of the main chain of tested compounds strongly influenced the organization and shape of the flower-like nanostructures formed, which were unique for 5/6-FAM-O2Oc-[Dap(GO2)]8-O2Oc-NH2 and typical for large proteins.


Assuntos
Permeabilidade da Membrana Celular/fisiologia , Ácidos Nucleicos/metabolismo , Polímeros/farmacologia , beta-Alanina/análogos & derivados , Linhagem Celular , Linhagem Celular Tumoral , Humanos , Nanoestruturas/química , Plasmídeos/metabolismo , Transfecção/métodos , beta-Alanina/farmacologia
16.
PLoS One ; 16(3): e0247595, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33780474

RESUMO

Children with Down syndrome (DS) are susceptible to two blood disorders, transient abnormal myelopoiesis (TAM) and Down syndrome-associated acute megakaryocytic leukemia (DS-AMKL). Mutations in GATA binding protein 1 (GATA1) have been identified as the cause of these diseases, and the expression levels of the resulting protein, short-form GATA1 (GATA1s), are known to correlate with the severity of TAM. On the other hand, despite the presence of GATA1 mutations in almost all cases of DS-AMKL, the incidence of DS-AMKL in TAM patients is inversely correlated with the expression of GATA1s. This discovery has required the need to clarify the role of GATA1s in generating the cells of origin linked to the risk of both diseases. Focusing on this point, we examined the characteristics of GATA1 mutant trisomy-21 pluripotent stem cells transfected with a doxycycline (Dox)-inducible GATA1s expression cassette in a stepwise hematopoietic differentiation protocol. We found that higher GATA1s expression significantly reduced commitment into the megakaryocytic lineage at the early hematopoietic progenitor cell (HPC) stage, but once committed, the effect was reversed in progenitor cells and acted to maintain the progenitors. These differentiation stage-dependent reversal effects were in contrast to the results of myeloid lineage, where GATA1s simply sustained and increased the number of immature myeloid cells. These results suggest that although GATA1 mutant cells cause the increase in myeloid and megakaryocytic progenitors regardless of the intensity of GATA1s expression, the pathways vary with the expression level. This study provides experimental support for the paradoxical clinical features of GATA1 mutations in the two diseases.


Assuntos
Síndrome de Down/sangue , Fator de Transcrição GATA1/metabolismo , Hematopoese/genética , Células-Tronco Embrionárias Humanas/metabolismo , Diferenciação Celular/genética , Linhagem Celular , Linhagem da Célula/genética , Síndrome de Down/genética , Doxiciclina/farmacologia , Fator de Transcrição GATA1/genética , Humanos , Leucemia Megacarioblástica Aguda/sangue , Leucemia Megacarioblástica Aguda/genética , Reação Leucemoide/sangue , Reação Leucemoide/genética , Megacariócitos/metabolismo , Células Mieloides/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Transfecção/métodos , Trissomia/genética
17.
PLoS One ; 16(3): e0243645, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33667217

RESUMO

Chemical transfection is broadly used to transiently transfect mammalian cells, although often associated with cellular stress and membrane instability, which imposes challenges for most cellular assays, including high-throughput (HT) assays. In the current study, we compared the effectiveness of calcium phosphate, FuGENE and Lipofectamine 3000 to transiently express two key voltage-gated ion channels critical in pain pathways, CaV2.2 and NaV1.7. The expression and function of these channels were validated using two HT platforms, the Fluorescence Imaging Plate Reader FLIPRTetra and the automated patch clamp QPatch 16X. We found that all transfection methods tested demonstrated similar effectiveness when applied to FLIPRTetra assays. Lipofectamine 3000-mediated transfection produced the largest peak currents for automated patch clamp QPatch assays. However, the FuGENE-mediated transfection was the most effective for QPatch assays as indicated by the superior number of cells displaying GΩ seal formation in whole-cell patch clamp configuration, medium to large peak currents, and higher rates of accomplished assays for both CaV2.2 and NaV1.7 channels. Our findings can facilitate the development of HT automated patch clamp assays for the discovery and characterization of novel analgesics and modulators of pain pathways, as well as assisting studies examining the pharmacology of mutated channels.


Assuntos
Canais de Cálcio Tipo N/genética , Canal de Sódio Disparado por Voltagem NAV1.7/genética , Transfecção/métodos , Potenciais de Ação/efeitos dos fármacos , Analgésicos/farmacologia , Animais , Células CHO , Canais de Cálcio Tipo N/metabolismo , Cricetinae , Cricetulus , Células HEK293 , Ensaios de Triagem em Larga Escala , Humanos , Microscopia de Fluorescência , Canal de Sódio Disparado por Voltagem NAV1.7/metabolismo , Dor/metabolismo , Dor/patologia , Técnicas de Patch-Clamp
18.
Nat Chem Biol ; 17(5): 593-600, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33686291

RESUMO

O-linked N-acetylglucosamine (O-GlcNAc) is an essential and dynamic post-translational modification that is presented on thousands of nucleocytoplasmic proteins. Interrogating the role of O-GlcNAc on a single target protein is crucial, yet challenging to perform in cells. Herein, we developed a nanobody-fused split O-GlcNAcase (OGA) as an O-GlcNAc eraser for selective deglycosylation of a target protein in cells. After systematic cellular optimization, we identified a split OGA with reduced inherent deglycosidase activity that selectively removed O-GlcNAc from the desired target protein when directed by a nanobody. We demonstrate the generality of the nanobody-fused split OGA using four nanobodies against five target proteins and use the system to study the impact of O-GlcNAc on the transcription factors c-Jun and c-Fos. The nanobody-directed O-GlcNAc eraser provides a new strategy for the functional evaluation and engineering of O-GlcNAc via the selective removal of O-GlcNAc from individual proteins directly in cells.


Assuntos
Antígenos de Neoplasias/metabolismo , Histona Acetiltransferases/metabolismo , Hialuronoglucosaminidase/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Glicoproteínas de Membrana/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Anticorpos de Domínio Único/química , Fator de Transcrição Sp1/metabolismo , Fatores de Transcrição/metabolismo , Antígenos de Neoplasias/química , Antígenos de Neoplasias/genética , Bioensaio , Domínio Catalítico , Sistemas de Liberação de Medicamentos/métodos , Expressão Gênica , Glicosilação , Células HEK293 , Histona Acetiltransferases/química , Histona Acetiltransferases/genética , Humanos , Hialuronoglucosaminidase/química , Hialuronoglucosaminidase/genética , Hidrólise , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Glicoproteínas de Membrana/genética , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Plasmídeos/química , Plasmídeos/metabolismo , Ligação Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Anticorpos de Domínio Único/metabolismo , Fator de Transcrição Sp1/genética , Fatores de Transcrição/genética , Transfecção/métodos
19.
Viruses ; 13(3)2021 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-33668972

RESUMO

The rotavirus species A (RVA) capsid contains the spike protein VP4, which interacts with VP6 and VP7 and is involved in cellular receptor binding. The capsid encloses the genome consisting of eleven dsRNA segments. Reassortment events can result in novel strains with changed properties. Using a plasmid-based reverse genetics system based on simian RVA strain SA11, we previously showed that the rescue of viable reassortants containing a heterologous VP4-encoding genome segment was strain-dependent. In order to unravel the reasons for the reassortment restrictions, we designed here a series of plasmids encoding chimeric VP4s. Exchange of the VP4 domains interacting with VP6 and VP7 was not sufficient for rescue of viable viruses. In contrast, the exchange of fragments encoding the receptor-binding region of VP4 resulted in virus rescue. All parent strains and the rescued reassortants replicated efficiently in MA-104 cells used for virus propagation. In contrast, replication in BSR T7/5 cells used for plasmid transfection was only efficient for the SA11 strain, whereas the rescued reassortants replicated slowly, and the parent strains failing to produce reassortants did not replicate. While future research in this area is necessary, replication in BSR T7/5 cells may be one factor that affects the rescue of RVAs.


Assuntos
Proteínas do Capsídeo/genética , Vírus Reordenados/genética , Infecções por Rotavirus/virologia , Rotavirus/genética , Capsídeo/fisiologia , Linhagem Celular , Humanos , Plasmídeos/genética , Genética Reversa/métodos , Transfecção/métodos , Replicação Viral/genética
20.
J Mater Chem B ; 9(9): 2275-2284, 2021 03 11.
Artigo em Inglês | MEDLINE | ID: mdl-33606863

RESUMO

MicroRNA (miRNA) based therapy for bone repair has shown promising results for regulating stem cell proliferation and differentiation, an efficient and stable vector for delivery of microRNA delivery is needed. The present study explored the stability and functionality of lyophilized mesoporous silica nanoparticles with core-cone structure and coated with polyethylenimine (MSN-CC-PEI) as a system for delivering Rattus norvegicus (rno)-miRNA-26a-5p into rat marrow mesenchymal cells (rBMSCs) to promote their osteogenic differentiation. We assessed the cellular uptake and transfection efficiency of nanoparticles loaded with labelled miRNA using confocal laser scanning microscopy and flow cytometry, and the cell viability using the MTT assay. The expression levels of osteogenic genes after one and two weeks were analysed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Extracellular matrix deposition and mineralization at 3 weeks were evaluated using Picro Sirius red and Alizarin red staining. We also assessed the performance of the delivery system after long term storage, by freeze drying rno-miRNA-26a-5p@MSN-CC-PEI with 5% trehalose and keeping them at -30 °C for 3 and 6 months. Osteogenic differentiation, matrix deposition, and mineralization were all significantly increased by rno-miRNA-26a-5p. In addition, this enhancement was not significantly altered by lyophilization and storage. Overall, these findings support the concept of MSN-CC-PEI as a delivery system for gene therapy. The complex of rno-miRNA-26a-5p@MSN-CC-PEI could efficiently transfect rBMSCs and enhance their osteogenic differentiation. In addition, the lyophilized complexes remain functional after 6 months of storage.


Assuntos
Diferenciação Celular/genética , Portadores de Fármacos/química , MicroRNAs/genética , Nanopartículas/química , Osteogênese/genética , Dióxido de Silício/química , Transfecção/métodos , Animais , Sobrevivência Celular , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , MicroRNAs/química , Polietilenoimina/química , Porosidade , Estabilidade de RNA , Ratos
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