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Characterization of the properties of seven promoters in the motor cortex of rats and monkeys after lentiviral vector-mediated gene transfer.

ABSTRACT: Lentiviral vectors deliver transgenes efficiently to a wide range of neuronal cell types in the mammalian central nervous system. To drive gene expression, internal promoters are essential; however, the in vivo properties of promoters, such as their cell type specificity and gene expression activity, are not well known, especially in the nonhuman primate brain. Here, the properties of five ubiquitous promoters (murine stem cell virus [MSCV], cytomegalovirus [CMV], CMV early enhancer/chicken ?-actin [CAG], human elongation factor-1? [EF-1?], and Rous sarcoma virus [RSV]) and two cell type-specific promoters (rat synapsin I and mouse ?-calcium/calmodulin-dependent protein kinase II [CaMKII?]) in rat and monkey motor cortices in vivo were characterized. Vesicular stomatitis virus G (VSV-G)-pseudotyped lentiviral vectors expressing enhanced green fluorescent protein (EGFP) under the control of the various promoters were prepared and injected into rat and monkey motor cortices. Immunohistochemical analysis revealed that all of the VSV-G-pseudotyped lentiviral vectors had strong endogenous neuronal tropisms in rat and monkey brains. Among the seven promoters, the CMV promoter showed modest expression in glial cells (9.4%) of the rat brain, whereas the five ubiquitous promoters (MSCV, CMV, CAG, EF-1?, and RSV) showed expression in glial cells (7.0-14.7%) in the monkey brain. Cell type-specific synapsin I and CaMKII? promoters showed excitatory neuron-specific expression in the monkey brain (synapsin I, 99.7%; CaMKII?, 100.0%), but their specificities for excitatory neurons were significantly lower in the rat brain (synapsin I, 94.6%; CaMKII?, 93.7%). These findings could be useful in basic and clinical neuroscience research for the design of vectors that efficiently deliver and express transgenes into rat and monkey brains.

SUBMITTER: Yaguchi M

PROVIDER: S-EPMC3869537 | biostudies-literature | 2013 Dec

REPOSITORIES: biostudies-literature

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Characterization of the properties of seven promoters in the motor cortex of rats and monkeys after lentiviral vector-mediated gene transfer.

Yaguchi Masae M Ohashi Yohei Y Tsubota Tadashi T Sato Ayana A Koyano Kenji W KW Wang Ningqun N Miyashita Yasushi Y

Human gene therapy methods 20131022 6

Lentiviral vectors deliver transgenes efficiently to a wide range of neuronal cell types in the mammalian central nervous system. To drive gene expression, internal promoters are essential; however, the in vivo properties of promoters, such as their cell type specificity and gene expression activity, are not well known, especially in the nonhuman primate brain. Here, the properties of five ubiquitous promoters (murine stem cell virus [MSCV], cytomegalovirus [CMV], CMV early enhancer/chicken β-ac ...[more]

PMID: 23964981

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Project description:The importance of retinal glial cells in the maintenance of retinal health and in retinal degenerations has not been fully explored. Several groups have suggested that secretion of neurotrophic proteins from the retina's primary glial cell type, the Müller cell, holds promise for treating retinal degenerations. Tight regulation of transgene expression in Müller cells is likely to be critical to the efficacy of long-term neuroprotective therapies, due to the genetic heterogeneity and progressive nature of retinal disease. To this end, we developed a modified lentiviral (LV) transfer vector (pFTMGW) to accelerate the testing and evaluation of novel transcriptional regulatory elements. This vector facilitates identification and characterization of regulatory elements in terms of size, cell specificity and ability to control transgene expression levels.A synthetic multiple cloning site (MCS) which can accept up to five directionally cloned DNA regulatory elements was inserted immediately upstream of an enhanced green fluorescent protein (eGFP) reporter. A cytomegalovirus (CMV) promoter, required for tat-independent viral packaging, is located around 2 kb upstream of the eGFP reporter and is capable of directing transgene expression. A synthetic transcription blocker (TB) was inserted to insulate the MCS/eGFP from the CMV promoter. We evaluated eGFP expression from pFTMGW and control constructs using flow cytometry and quantitative reverse transcriptase polymerase chain reaction (RT-PCR). We also tested and compared the activity and cell specificity of a computationally identified promoter fragment from the rat vimentin gene (Vim409) in transfection and lentiviral infection experiments using fluorescence microscopy.Transfection data, quantitative RT-PCR, and flow cytometry show that around 85% of expression from the CMV promoter was blocked by the TB element, allowing direct evaluation of expression from the Vim409 candidate promoter cloned into the MCS. Lentiviruses generated from this construct containing the Vim409 promoter (without the TB element) drove robust eGFP expression in Müller cells in vitro and in vivo.The TB element efficiently prevented eGFP expression by the upstream CMV promoter and the novel MCS facilitated testing of an evolutionarily conserved regulatory element. Additional sites allow for combinatorial testing of additional promoter, enhancer, and/or repressor elements in various configurations. This modified LV transfer vector is an effective tool for expediting functional analysis of gene regulatory elements in Müller glia, and should prove useful for promoter analyses in other cell types and tissues.

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Dataset Information

Characterization of the properties of seven promoters in the motor cortex of rats and monkeys after lentiviral vector-mediated gene transfer.

Publications

Characterization of the properties of seven promoters in the motor cortex of rats and monkeys after lentiviral vector-mediated gene transfer.

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