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Transgenic animal

a technology of transgenic animals and knockouts, applied in the field of transgenic animals, can solve the problems that the phenotypic effect of a simple knockout fails to provide useful or accurate information about the function of the gene, and achieve the effect of modulating gpcr signalling and inhibiting rgs activity

Inactive Publication Date: 2008-07-10
WYETH LLC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0011]The present invention further includes a method for making a transgenic animal that includes the steps of identifying a variant coding sequence controlling a dominant negative function and introducing the variant coding sequence into animal cells to produce transgenic animal cells that replicate to produce a transgenic animal. The method can also include the step of providing a tissue-specific expression control element operationally linked to the variant coding sequence so that expression of the dominant negative function is confined to a desired tissue type, such as the brain, of the transgenic animal. The method can produce many animal lines bearing the same transgene but differing in transgene expression levels. Comparing the varied phenotypes of such lines permits analysis of the quantitative influence of the dominant negative transgene.

Problems solved by technology

In the latter case, the related genes may interact in a hierarchical or compensatory manner such that the phenotypic effect of a simple knockout fails to provide useful or accurate information of the gene's function.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

example 1

Cloning of the Transgene PCR

Amplification and Cloning of The Gαq G188S Transgene

[0078]Plasmid pcDNAamp Gqsst (DiBello et al. J. Biol. Chem. 1998, 273:5780-5784) was used as a template to PCR a 1.1 kb fragment of the mouse Gαq cDNA. Gqsst contains a G to A conversion at nucleotide 573 and a G to C conversion at nucleotide 575 that converts a glycine residue to serine at amino acid residue 188 (G188S) (see FIG. 1). In addition, several nucleotide changes were incorporated 5′ to the mutation to convert an endogenous Gαq amino acid sequence portion to that of an EE epitope tag (FIG. 1) to allow mutant Gαq protein detection using anti-EE antisera. “EE” refers to a two glutamate sequence that is part of an epitope recognized by a commercially available mouse monoclonal antibody Glu-Glu (Babco, Berkley Antibody Company, Richmond, Calif.). This antibody was raised against the sequence CEEEEYMPE and is specific for either six amino acid sequences EYMPME or EFMPME. The EE epitope is known to ...

example 2

Construction of Tissue Specific Transgene

Expression Construct Cloning of Thy1.2-Gαq (G188S)

[0079]Plasmid Gαq (G188S)-GemT was cut with Xho I and the 1.1 kb Gαq cDNA was excised from a 1% agarose gel and cluted by Gene-Clean (Bio-101) according to manufacturer's protocol. Plasmid Thy1.2 containing a 6.7 kb NotI-PvuI mouse Thy1.2 minigene cloned in pUC19 was described previously. The 1.1 kb Gαq DNA was ligated to Thy1.2 vector DNA cut with Xho I and treated with calf intestinal alkaline phosphatase to remove 5′ phosphate ends (Sambrook et al., 1982). Clone Thy1.2-Gαq (G188S) containing the 1.1 kb mutant Gαq cDNA in the correct orientation in the XhoI cloning site of the Thy1.2 expression cassette was verified by restriction mapping as well as DNA sequence analysis (see FIG. 2).

example 3

Preparation of Transgenic Rat

Transgene Preparation For DNA Microinjection

[0080]Construct Thy1.2-Gαq G188S DNA (100 micrograms) was cut with NotI and PvuI restriction enzymes overnight at 37° C. The DNA was electrophoresed on 1% low melting point agarose gel (BRL) to allow separation of the 7.8 kb Thy1.2-Gαq G188S transgene DNA from smaller sized 2 kb and 1 kb fragments generated from restriction of the pUC19 vector (New England Biolabs) backbone. The 7.8 kb fragment was excised from the LMP gel and heated to 70° C. to melt the agarose. Once liquefied, an equal volume of phenol was used to extract the gel mix. The aqueous portion was re-extracted using a phenol-chloroform mix followed by chloroform. The aqueous fraction was then precipitated using 2 volumes of 95% ethanol, followed by a 70% ethanol wash. The DNA pellet was resuspended in 2.5 ml TE buffer. One gram of cesium chloride (Sigma, St. Louis, Mo.) was added for every 1 ml of DNA-TE mix and gently dissolved. Ethidium bromide ...

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Abstract

A transgenic rat containing in its genome a nucleotide sequence encoding a Ga subunit protein, which Ga protein subunit is uncoupled from regulation by Regulators of G-Protein Signaling (RGS) proteins, which Gx subunit protein is eventually the dominant-negative G188S mutant of Gax9, which nucleotide sequence is operatively associated with a neuron-specific expression control sequence, wherein the transgenic rat expresses the GA subunit protein in neural cells resulting in extended D-protein coupled receptor signaling mediated by the Ga subunit protein.

Description

[0001]This application claims priority under 35 U.S.C. § 119 from Provisional Application Nos. 60 / 199,209 and 60 / 245,473 filed Apr. 24, 2000 and Nov. 3, 2000, respectively, which are each incorporated herein by reference in their entirety.FIELD OF THE INVENTION[0002]The invention generally relates to transgenic animals (rats and mice) and to animal models of human disease. The invention particularly relates to transgenic animals which can serve as animal models of human diseases or conditions modulated by proteins that regulate G-protein signaling.BACKGROUND OF THE INVENTION[0003]The RGS (Regulators of G-protein Signaling) proteins act to desensitize G protein mediated signal transduction by accelerating the endogenous GTPase activity of activated Gα subunits. RGS proteins have been demonstrated to function as GAPs (GTPase accelerating proteins) for Gαo, Gαi, Gαz and Gαq subtypes of the Gα subunit (Grafstein-Dunn, et al. Mol. Brain. Res. 2001, 88:113-123; for a review see Hepler, Tr...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): G01N33/53A01K67/027C07K14/47C12N15/09C12N15/85
CPCA01K67/0275A01K2217/05A01K2227/105A01K2267/03C12N2830/85C07K14/4722C12N15/8509C12N2830/008A01K2267/0356
Inventor YOUNG, KATHLEENHOWLAND, DAVID S.MARQUIS, KAREN L.ROSENZWEIG-LIPSON, SHARONCOCKETT, MARK IAN
Owner WYETH LLC
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