RESUMO
Nucleic acid-based therapies hold great promise for treatment of skin disorders if delivery challenges can be overcome. To investigate one mechanism of nucleic acid delivery to keratinocytes, a fixed mass of expression plasmid was intradermally injected into mouse footpads in different volumes, and reporter expression was monitored by intravital imaging or skin sectioning. Reporter gene expression increased with higher delivery volumes, suggesting that pressure drives nucleic acid uptake into cells after intradermal injections similar to previously published studies for muscle and liver. For spatiotemporal analysis of reporter gene expression, a dual-axis confocal (DAC) fluorescence microscope was used for intravital imaging following intradermal injections. Individual keratinocytes expressing hMGFP were readily visualized in vivo and initially appeared to preferentially express in the stratum granulosum and subsequently migrate to the stratum corneum over time. Fluorescence microscopy of frozen skin sections confirmed the patterns observed by intravital imaging. Intravital imaging with the DAC microscope is a noninvasive method for probing spatiotemporal control of gene expression and should facilitate development and testing of new nucleic acid delivery technologies.
Assuntos
DNA/administração & dosagem , Regiões Promotoras Genéticas , Pele/metabolismo , Animais , DNA/metabolismo , Epiderme/metabolismo , Feminino , Genes Reporter , Terapia Genética/métodos , Injeções Intradérmicas , Queratinócitos/metabolismo , Camundongos , Microscopia de Fluorescência , Plasmídeos/genética , Plasmídeos/metabolismo , PressãoRESUMO
Small interfering RNAs (siRNAs) can be designed to specifically and potently target and silence a mutant allele, with little or no effect on the corresponding wild-type allele expression, presenting an opportunity for therapeutic intervention. Although several siRNAs have entered clinical trials, the development of siRNA therapeutics as a new drug class will require the development of improved delivery technologies. In this study, a reporter mouse model (transgenic click beetle luciferase/humanized monster green fluorescent protein) was developed to enable the study of siRNA delivery to skin; in this transgenic mouse, green fluorescent protein reporter gene expression is confined to the epidermis. Intradermal injection of siRNAs targeting the reporter gene resulted in marked reduction of green fluorescent protein expression in the localized treatment areas as measured by histology, real-time quantitative polymerase chain reaction and intravital imaging using a dual-axes confocal fluorescence microscope. These results indicate that this transgenic mouse skin model, coupled with in vivo imaging, will be useful for development of efficient and 'patient-friendly' siRNA delivery techniques and should facilitate the translation of siRNA-based therapeutics to the clinic for treatment of skin disorders.
Assuntos
Proteínas de Fluorescência Verde/genética , Queratinócitos/metabolismo , Camundongos Transgênicos , Interferência de RNA , RNA Interferente Pequeno/administração & dosagem , Pele/metabolismo , Animais , Genes Reporter , Humanos , Luciferases/genética , Camundongos , Modelos AnimaisRESUMO
To gain more insight about Escherichia coli tmRNA structure, NiCR, a square planar macrocyclic nickel (II) complex, was used to probe guanine N7 exposure. On the basis of this additional structural information, a refined secondary structure of the molecule is proposed. In addition to its known specificity for guanine N7, we show here that the chemical probe can also cleave at specific uridine residues. In contrast to the alkaline-labile modification of guanine, the reactivity of NiCR at these uridine residues results in direct strand scission. To better characterize the uridine cleavage sites and assess the importance of the RNA structure for the reaction to occur, smaller RNA molecules derived from one pseudoknot (PK4) of E. coli tmRNA containing two uridine cleavage sites were engineered and probed. It is shown that this pseudoknot can fold by itself in solution and that the expected uridine residues are also cleaved by the nickel complex, suggesting that only a local sequence and/or structural context is required for cleavage. In E. coli tmRNA, the five uridine cleavage sites are located in double-stranded regions. These sites contain a G-U wobble base-pair and a downstream uridine which is cleaved. Using smaller RNAs derived from one stem of PK4, systematic changes in the proposed recognition motif indicate that the G-U pair is required for cleavage. Furthermore, there is no cleavage if the G-U pair is reversed. If the recognition motif is moved within the stem, the cleavage site moves accordingly. Additionally, if the recognition motif is changed such that the G-U pair is flanked by two uridine residues, the reactivity occurs only at the 3' uridine. Radical quenching studies have indicated that sulfate radical, as in the case of guanine oxidation, is involved in uridine oxidation. Although additional studies are required to better characterize the reaction, this paper reports a novel specificity for a chemical probe which may be useful for investigating structural motifs involving G-U pairs in folded RNAs.
Assuntos
Escherichia coli/química , Conformação de Ácido Nucleico , Compostos Organometálicos/metabolismo , RNA Bacteriano/química , Uridina/metabolismo , Composição de Bases/genética , Sequência de Bases , Radicais Livres/metabolismo , Guanina/metabolismo , Magnésio/farmacologia , Sondas Moleculares , Dados de Sequência Molecular , Mutação/genética , Níquel/química , Oligorribonucleotídeos/metabolismo , Compostos Organometálicos/química , Ésteres do Ácido Sulfúrico/metabolismoRESUMO
Two gel electrophoretic methods are described for detection of 7, 8-dihydro-8-oxoguanine and 7,8-dihydro-8-oxoadenine based on their further oxidation with one-electron oxidants including IrCl62-and IrBr62-. The products of nucleobase oxidation lead to enhanced piperidine-sensitive cleavage and to highly visible stop points in a primer extension assay. 8-oxoG and 8-oxoA lesions may be distinguished by the latter's inability to be oxidized by IrBr62-compared to IrCl62-Comparison is also made to oxidation by MnO4-.