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Transgenic non-human organisms with non-functional TSPO genes

A non-functional, non-human technology, applied in the direction of organic active ingredients, neurotransmitter receptors, animal/human proteins, etc., can solve the problem of expansion

Inactive Publication Date: 2016-01-27
AUSTRALIAN NUCLEAR SCI & TECH ORGANISAT +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Furthermore, animals in which at least one allele of the TSPO gene is non-functional or absent will significantly expand the currently limited possibilities for studying the regulation of other TSPO-dependent biological pathways

Method used

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  • Transgenic non-human organisms with non-functional TSPO genes
  • Transgenic non-human organisms with non-functional TSPO genes
  • Transgenic non-human organisms with non-functional TSPO genes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0391] Example 1: Development of TSPO knockout animals

[0392] Materials and methods

[0393] Construct design and transgenic animal production

[0394] Development of TSPO knockout mice was performed by Ozgene (Bentley DC, WA, Australia). TSPO knockout mice were generated using the Cre-Lox recombination method. To generate TSPO knockout mice, targeting constructs were generated with some added homology to the wild-type TSPO allele (see figure 1 ). The construct also contained a pair of LoxP sites flanking exons 2 and 3, which contained the TSPO initiation codon, a neomycin cassette to select for the targeting construct, and a flippase recognition target (FRT) successful acquisition of the site. An FRT site was included to allow generation of conditional knockout mice at a later stage. The neomycin cassette confers neomycin resistance to cells that successfully incorporate the construct. The construct was delivered by electroporation into Bruce4 mouse embryonic stem (E...

Embodiment 2

[0419] Example 2: Radioligand Membrane Binding

[0420] Materials and methods

[0421] Membrane preparation

[0422] Tissue samples were homogenized with a T25 digital Ultra-Turrax homogenizer (Ika, Wilmington, NC, USA) in approximately 45 mL of ice-cold TRIS buffer (pH 7.4) at a speed setting of 5 or 20000 rpm, by centrifugation at 48000 g Collect, then discard the supernatant. This step is then immediately repeated as an additional wash step to remove any soluble interfering substances bound by the radioligand (Byland et al. 1993). After a second centrifugation and removal of the supernatant, the samples were resuspended in approximately 50 volumes of ice-cold TRIS buffer (pH 7.4). Aliquots of samples were stored at -80°C until use.

[0423] Protein Concentration Measurement

[0424] Protein concentrations were measured using the bicinchoninic acid (BCA) protein assay kit (ThermoFisher Scientific, Scoresby, VIC, Australia) following the supplier's instructions. Briefly...

Embodiment 3

[0432] Example 3: Analysis of TSPO levels in tissues by autoradiography

[0433] Materials and methods

[0434] Snap-frozen tissues were sectioned at 20 μm in a cryostat, thaw-mounted on poly-L-lysine-coated slides, and stored at −80° C. until the day of the experiment. On the day of the experiment, slides were thawed at room temperature and air-dried with a cold stream. Total and non-specific binding was determined by incubation with 1 nM 3H-PK11195 at room temperature for 20 minutes in the presence or absence of 3 μM of PK11195 in 130 mM TRIS-HCl buffer (pH 7.4). After incubation, slides were briefly submerged twice in 130 mM TRIS-HCl buffer and washed twice in fresh 130 mM TRIS-HCl for 5 minutes at room temperature. Finally, place the slide in cold distilled water (H 2 Rinse briefly 3 times in O), air dry under a cold stream, and allow to dry overnight. Sections were exposed to Kodak BioMaxMR film as well as trace amounts of tritium with known activity concentrations in...

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Abstract

The present invention relates to transgenic animal models. Specifically, the present invention relates to transgenic animal models for applications associated with TSPO-related normal physiology, diseases and disorders. The present invention features transgenic nonhuman animal comprising cells with at least one copy of a non-functional, endogenous TSPO gene. Also disclosed are compounds for investigating or modulating TSPO-related functions.

Description

[0001] cross reference [0002] This application claims priority to Australian Provisional Patent Application 2013900858 filed 13 March 2013, 2013903696 filed 25 September 2013 and 2013905101 filed 24 December 2013 , all of which are fully incorporated herein by cross-reference. technical field [0003] The present invention relates to transgenic animal models. In particular, the present invention relates to transgenic animal models for applications related to TSPO-related normal physiology, diseases and disorders. Background of the invention [0004] Translocator protein (TSPO), previously known as peripheral benzodiazepine receptor (PBR), is an 18 kDa membrane protein mainly localized on the outer mitochondrial membrane and widely distributed throughout the body. Its expression level is different in different tissues and organs. In particular, healthy adults have very low levels of TSPO in the brain parenchyma, while the kidneys, lungs and heart express high levels of T...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85A01K67/027C12N5/12A61P3/00A61P25/00A61P35/00A61P25/28A61K31/437C07D471/04
CPCA61K31/437C07D471/04C07K14/70571A01K2227/105A01K2267/0312A01K2267/0318A01K67/0276A61P25/00A61P25/28A61P3/00A61P3/04A61P35/00A61P43/00G01N33/5088
Inventor 瑞安·米德尔顿理查德·巴纳蒂刘国军
Owner AUSTRALIAN NUCLEAR SCI & TECH ORGANISAT
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