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Nav1.7 knockout mice and uses thereof

A technology of nav1.7 and mice, which is applied in the direction of recombinant DNA technology, preparations for in vivo experiments, and the use of vectors to introduce foreign genetic materials, etc., can solve problems such as feeding failure

Inactive Publication Date: 2014-01-01
AMGEN INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0014] Nassar et al. used gene ablation in mice to examine Na V 1.7 functions in pain pathways; however, they report finding (unlike humans) a global Na V 1.7-Null mutants die shortly after birth, apparently as a result of feeding failure

Method used

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  • Nav1.7 knockout mice and uses thereof
  • Nav1.7 knockout mice and uses thereof
  • Nav1.7 knockout mice and uses thereof

Examples

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Embodiment approach

[0101] Included within the scope of the present invention are global Nav1.7 KO mice in which they have been "knocked out" or "knocked out" by insertion of a gene from the mouse (which may have a modified nucleotide sequence) or a transgene into" 1, 2 or more additional genes of interest. Such mammals can be created by repeating the methods set forth herein for generating each "knockout" or transgenic "knockin" construct or by breeding mammals each with a single gene knockout with each other, followed by screening for the presence of two knockouts. Mammals of one or more knockout and / or knockin genotypes are generated. The gene to be knocked out or knocked in can be any gene as long as at least some sequence information about the DNA to be disrupted or recombinantly expressed is available for use in making constructs and screening probes.

[0102] Knockout gene selection

[0103] Typically, the DNA to be used in a knockout construct will be one or more exon and / or intron re...

Embodiment 1

[0234] Example 1 .Backcross and outcross

[0235] Due to the reported Na V 1.7 KO animals (heterozygous Nav1.7 from Deltagen backcrossed with C57BL / 6 for at least 8 generations, San Mateo, CA: B6.129P2-Scn9atm1Dgen / J + / - mouse) (Nassar et al., 2004), we outcrossed these animals to a CD1 background to increase the vigor of the strain, and backcrossed them to the BALB / c strain respectively to generate isogenic strains ( The first breeding pair obtained in May 2011). (see, Figure 1A -B).

[0236] No significant differences were observed in Scn9a-CD1KO or Scn9a-Balbc KO animals. We call these animals Na V 1.7 KO, and specify the strain if required. All data shown in this article were collected from a single outcross and backcross. Further backcrosses (for the BALB / c line only) are currently underway and the results obtained so far are consistent across the N4 backcross generation. No further increase in survival was observed. The final outcome of the backcross is still t...

Embodiment 2

[0259] Example 2 : Preparation of artificial mouse milk (AMA)

[0260] The method and composition of artificial mouse milk prepared was modeled closely following the protocols described by Yajima and coworkers and by Auestad and coworkers. (Yajima, M, et al., A Chemically Derived Milk Substitute that is Compatible with Mouse Milk for Artificial Rearing of Mouse Pups'Exp.Amin.55(4), 391-397, (2006); Auestad, N, et al., Milk-substitutes comparable to rat's milk; their preparation, composition and impact on development and metabolism in the artificially reared rat, Br.J.Nutr.61:495-518(1989)).

[0261] Reagents and methods. Reagents used, including sources, amounts used, solvents and amounts used, and various notes are listed in Table 3 (below).

[0262] method. Step 1: Weigh the sodium hydroxide and transfer it to a 10-L beaker. Distilled water (1.3 L) was added and an overhead stirrer was installed. Potassium hydroxide was weighed and added to this solution, and the sa...

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Abstract

A viable global NaV1.7− / − knockout mouse is disclosed, and a breeding colony of global NaV1.7− / − knockout mice. Also disclosed are an isolated mouse gamete that does not encode a functional NaV1.7− / −, produced by the NaV1.7− / − knockout mouse; an isolated NaV1.7− / − mouse cell, or a progeny cell thereof, isolated from the NaV1.7− / − knockout mouse; and a primary cell culture or a secondary cell line and a tissue or organ explant or culture thereof derived from the NaV1.7− / − knockout mouse. Disclosed also are a hybridoma, wherein the hybridoma was originally formed from the fusion of the isolated NaV1.7− / − mouse cell mouse cell and a myeloma cell, and a method of making an antibody. Also disclosed are assays useful for screening prospective NaV1.7 inhibitors and dose ranging a test NaV17 inhibitor compound, which were validated using the NaV1.7− / − knockout mouse.

Description

field of invention [0001] This application includes the Sequence Listing in ASCII "text" for use in computer readable form (CRF) and paper copies as required by 37 C.F.R. Sections 1.821(c) and 1.821(e), and is hereby incorporated by reference in its entirety This article. The name of the "text" file created on January 17, 2012 is: A-1588-WO-PCT-SeqList011812.ST25.txt and the size is 2kb. [0002] Throughout this application, various publications are referenced within parentheses or parentheses. The disclosures of these publications in their entirety are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains. Background of the invention [0003] Discussion of related technologies [0004] While the use of isolated cell lines (i.e., in vitro systems) is useful for understanding the physiological roles of various genes and the proteins they produce, studies of the effects of such proteins can be do...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85A01K67/027C07K16/00A61K49/00G01N33/50
CPCA01K2217/077A61K49/0008A01K2227/105C07K16/00A01K67/0276A01K2267/03C12N15/8509A01K67/027C12N5/0609C12N5/061
Inventor J.金格拉斯S.I.麦唐诺
Owner AMGEN INC
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