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High-level expression by tissue/cancer-specific promoter with strict specificity using a single-adenoviral vector.

ABSTRACT: Tissue-/cancer-specific promoters for use in adenovirus vectors (AdVs) are valuable for elucidating specific gene functions and for use in gene therapy. However, low activity, non-specific expression and size limitations in the vector are always problems. Here, we developed a 'double-unit' AdV containing the Cre gene under the control of an ?-fetoprotein promoter near the right end of its genome and bearing a compact 'excisional-expression' unit consisting of a target cDNA 'upstream' of a potent promoter between two loxPs near the left end of its genome. When Cre was expressed, the expression unit was excised as a circular molecule and strongly expressed. Undesired leak expression of Cre during virus preparation was completely suppressed by a dominant-negative Cre and a short-hairpin RNA against Cre. Using this novel construct, a very strict specificity was maintained while achieving a 40- to 90-fold higher expression level, compared with that attainable using a direct specific promoter. Therefore, the 'double-unit' AdV enabled us to produce a tissue-/cancer-specific promoter in an AdV with a high expression level and strict specificity.

SUBMITTER: Kanegae Y

PROVIDER: S-EPMC3025582 | biostudies-other | 2011 Jan

REPOSITORIES: biostudies-other

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High-level expression by tissue/cancer-specific promoter with strict specificity using a single-adenoviral vector.

Kanegae Yumi Y Terashima Miho M Kondo Saki S Fukuda Hiromitsu H Maekawa Aya A Pei Zheng Z Saito Izumu I

Nucleic acids research 20101104 2

Tissue-/cancer-specific promoters for use in adenovirus vectors (AdVs) are valuable for elucidating specific gene functions and for use in gene therapy. However, low activity, non-specific expression and size limitations in the vector are always problems. Here, we developed a 'double-unit' AdV containing the Cre gene under the control of an α-fetoprotein promoter near the right end of its genome and bearing a compact 'excisional-expression' unit consisting of a target cDNA 'upstream' of a potent ...[more]

PMID: 21051352

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Project description:Visualization of neurons is indispensable for the investigation of neuronal circuits in the central nervous system. Virus vectors have been widely used for labeling particular subsets of neurons, and the adeno-associated virus (AAV) vector has gained popularity as a tool for gene transfer. Here, we developed a single AAV vector Tet-Off platform, AAV-SynTetOff, to improve the gene-transduction efficiency, specifically in neurons. The platform is composed of regulator and response elements in a single AAV genome. After infection of Neuro-2a cells with the AAV-SynTetOff vector, the transduction efficiency of green fluorescent protein (GFP) was increased by approximately 2- and 15-fold relative to the conventional AAV vector with the human cytomegalovirus (CMV) or human synapsin I (SYN) promoter, respectively. We then injected the AAV vectors into the mouse neostriatum. GFP expression in the neostriatal neurons infected with the AAV-SynTetOff vector was approximately 40-times higher than that with the CMV or SYN promoter. By adding a membrane-targeting signal to GFP, the axon fibers of neostriatal neurons were clearly visualized. In contrast, by attaching somatodendritic membrane-targeting signals to GFP, axon fiber labeling was mostly suppressed. Furthermore, we prepared the AAV-SynTetOff vector, which simultaneously expressed somatodendritic membrane-targeted GFP and membrane-targeted red fluorescent protein (RFP). After injection of the vector into the neostriatum, the cell bodies and dendrites of neostriatal neurons were labeled with both GFP and RFP, whereas the axons in the projection sites were labeled only with RFP. Finally, we applied this vector to vasoactive intestinal polypeptide-positive (VIP+) neocortical neurons, one of the subclasses of inhibitory neurons in the neocortex, in layer 2/3 of the mouse primary somatosensory cortex. The results revealed the differential distribution of the somatodendritic and axonal structures at the population level. The AAV-SynTetOff vector developed in the present study exhibits strong fluorescence labeling and has promising applications in neuronal imaging.

Project description:Although techniques for cell-specific gene expression via viral transfer have advanced, many challenges (e.g., viral vector design, transduction of genes into specific target cells) still remain. We investigated a novel, simple methodology for using adenovirus transfer to target specific cells of the kidney tubules for the expression of exogenous proteins. We selected genes encoding sodium-dependent phosphate transporter type 2a (NPT2a) in the proximal tubule, sodium-potassium-2-chloride cotransporter (NKCC2) in the thick ascending limb of Henle (TALH), and aquaporin 2 (AQP2) in the collecting duct. The promoters of the three genes were linked to a GFP-coding fragment, the final constructs were then incorporated into an adenovirus vector, and this was then used to generate gene-manipulated viruses. After flushing circulating blood, viruses were directly injected into the renal arteries of rats and were allowed to site-specifically expression in tubule cells, and rats were then euthanized to obtain kidney tissues for immunohistochemistry. Double staining with adenovirus-derived EGFP and endogenous proteins were examined to verify orthotopic expression, i.e. "adenovirus driven NPT2a-EGFP and endogenous NHE3 protein", "adenovirus driven NKCC2-EGFP and endogenous NKCC2 protein" and "adenovirus driven AQP2-EGFP and endogenous AQP2 protein". Owing to a lack of finding good working anti-NPT2a antibody, an antibody against a different protein (sodium-hydrogen exchanger 3 or NHE3) that is also specifically expressed in the proximal tubule was used. Kidney structures were well-preserved, and other organ tissues did not show EGFP staining. Our gene transfer method is easier than using genetically engineered animals, and it confers the advantage of allowing the manipulation of gene transfer after birth. This is the first method to successfully target gene expression to specific cells in the kidney tubules. This study may serve as the first step for safe and effective gene therapy in the kidney tubule diseases.

Dataset Information

High-level expression by tissue/cancer-specific promoter with strict specificity using a single-adenoviral vector.

Publications

High-level expression by tissue/cancer-specific promoter with strict specificity using a single-adenoviral vector.

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