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Somatotransgenic bioimaging

A technology for transgenic animals and gene products, which can be used in genetic engineering, biochemical equipment and methods, and the determination/inspection of microorganisms, which can solve problems such as limitations and time-consuming

Inactive Publication Date: 2010-03-24
UCL BUSINESS PLC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Peripheral blood sampling and biochemical analysis are time-consuming and limited by animal protocols

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0110] Experiments were performed to determine the possibility of achieving long-term tissue-specific transgene expression in mice.

[0111] Vector Preparation and Validation

[0112] gp64-pseudotypes for long-term analysis were prepared as described by Seppen et al. luciferase vector and vsvg-pseudotyped luciferase vector.

[0113] The lentivirus was prepared as follows: Inoculate 2 × 10 7 293T production cells. The following day, the following amounts of plasmid DNA were mixed in OptiMEM (Invitrogen, Paisley, UK) in each T-150 flask to a final volume of 5 ml: vector construct (pHR.SINcpptSEW) 40 μg, pMDG.2 / pHCMVwhvGP64 10 μg, pCMVΔ8.7430 μg. Polyethylenimine (PEI, 25 kDa) (Sigma, Poole, UK) was added to 5 ml OptiMEM to a final concentration of 2 μM and filtered through a 0.22 μm filter. The DNA was added dropwise to the PEI solution and incubated at room temperature for 20 minutes. The DNA / PEI solution was added to the 293T cells and incubated at 37°C, 5% CO 2 Incuba...

Embodiment 2

[0122] To assess long-term transgene expression, a single dose of gp64 / HIV luciferase (approximately 3 × 10 7 iu). Mice were biologically imaged over a year and older and luciferase bioluminescence was compared to controls (n=2). In vivo luciferase bioimaging was performed as described in Example 1. Luciferase expression was significantly higher than control throughout the analysis and persisted throughout the study ( Figure 6 ). The results demonstrate that overt expression can be detected for up to one year after dosing.

Embodiment 3

[0124] Genetic bioeffectors useful in animal models were tested in vitro (Examples 3-5).

[0125] NIH-3T3 cells were transfected with a plasmid containing a TGF-β response element driving luciferase expression. These cells were then transduced with a retroviral vector expressing TGF-β3. The SBE4 response element is specific for TGF-β activation through smad2 / 3-mediated transcriptional activation. This Smad activation can be further characterized as Smad2-specific transcriptional activation using ARE response elements together with the Xenopus Fast-1 trans-effector (a single ARE is specific for Samd2 / 3 only). The BMP-specific response elements are activated by activation of Smad1 / 5 / 8 and should not respond to activation of TGF-β3. Finally, Samd7 is a suppressor Smad known to be upregulated in a negative feedback loop to TGF-β3 activation. The experiment was carried out as follows:

[0126] Press 1×10 6Cells / well were prepopulated with NIH-3T3 cells and transfected with 10 ...

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Abstract

The invention relates to modelling diseases, to screening for compounds that modulate such diseases and to assaying drug metabolism and toxicity in non-human transgenic animals, by a novel technique developed by the inventors known as ''somatotransgenic bioimaging''. The invention thus provides: a method for determining whether the expression of a reporter gene is modulated by a compound or a method of evaluating the metabolism and / or toxicity of a compound, said method comprising: (a) administering said compound to a non-human transgenic animal, generated by gene transduction of one or more specific tissues when in utero or neonatal, with a vector comprising a bioluminescent reporter gene operably linked to a genetic element responsive to a pathology or therapy or to a genetic element responsive to drug metabolism and / toxicity; and (b) determining whether or not of said compound has an effect on the expression of said reporter gene in said specific tissue or tissues and / or determiningthe extent of any such effect, said determination comprising detecting from the animal bioluminescence caused by the activity of the gene product of the reporter gene. In some embodiments cells pre-transduced with vectors of the invention may also be introduced into the animals instead of delivering the vectors directly.

Description

technical field [0001] The present invention involves modeling lesions in non-human transgenic animals, screening for compounds that modulate these lesions, and assessing drug metabolism and toxicity through a new technology called "somatotransgenic bioimaging." Background technique [0002] drug validation [0003] Identification of potential therapeutics typically begins with the use of high-throughput in vitro techniques. Successful candidate compounds then need to be validated in vivo, such as in rodent models, followed by full-scale primate trials or clinical trials. [0004] Traditional drug trials rely on measurements in peripheral, secreted or excreted body fluids or biopsies, and often rely on endpoint analyses. Measurements made on body fluids or tissues rely on suitable experimental variables, ie they only function when there is a substance in the body fluid or tissue that changes in response to the administration of the candidate compound. Endpoint analysis re...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/63
CPCA01K2267/0393A01K2267/0337C12Q1/6897C12N15/8509A01K2267/035
Inventor S·N·沃丁顿T·R·麦凯
Owner UCL BUSINESS PLC
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