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Nonhuman model animal unresponsive to immunopotentiating synthetic compound achieved

Foreign code F110005238
File No. B02-01US
Posted date Aug 29, 2011
Country United States of America
Application number 49650102
Gazette No. 20050235372
Gazette No. 7608750
Date of filing Nov 22, 2002
Gazette Date Oct 20, 2005
Gazette Date Oct 27, 2009
International application number JP2002012234
International publication number WO2003043588
Date of international filing Nov 22, 2002
Date of international publication May 30, 2003
Priority data
  • P2001-358295 (Nov 22, 2001) JP
  • 2002WO-JP12234 (Nov 22, 2002) WO
Title Nonhuman model animal unresponsive to immunopotentiating synthetic compound achieved
Abstract (US7608750)
The present invention relates to provide a non-human animal model unresponsive to a synthetic compound wherein a gene function encoding TLR7 that recognizes an immunopotentiating synthetic compound such as imidazoquinoline lacks on is genomic locus.
Whole or part of a gene fragment of a gene site including an intracellular region and a transmembrane region of a TLR7 gene obtained from a mouse gene library is replaced by a plasmid including poly A signal and a marker gene to construct a targeting vector.
Then, this targeting vector is linearized and transferred into embryonic stem cells.
The target embryonic stem cells wherein the TLR7 gene function is deleted are microinjected into a mouse blastocyst to generate a chimeric mouse.
Then, this chimeric mouse is crossed with a wild-type mouse to generate a heterozygote mouse.
Next, the heterozygote mice are intercrossed to obtain a TLR7 knockout mouse.
Scope of claims [claim1]
1. A Toll-like receptor (TLR)7 homozygous knockout mouse whose genome comprises a disruption of a TLR7 gene, wherein the TLR7 homozygous knockout mouse lacks an increase of interferon-alpha compared with a wild-type mouse when R-848 is intraperitoneally injected into the mouse, and wherein a gene function that encodes TLR7 is deficient on its genomic locus.
[claim2]
2. The Toll-like receptor (TLR)7 homozygous knockout mouse according to claim 1, which is obtained by the following steps: (i) constructing a targeting vector by replacing whole or a part of a gene fragment of a gene site including an intracellular region and transmembrane region of a TLR7 gene obtained by screening a mouse gene library with a plasmid including a poly A signal and a marker gene; (ii) linearizing the targeting vector; (iii) introducing the linearized targeting vector into a mouse embryonic stem cell; (iv) microinjecting the targeted embryonic stem cell whose TLR7 gene function is deficient into a blastocyst of a mouse to generate a chimeric mouse; (v) intercrossing the chimeric mouse with a wild-type mouse to generate a heterozygote mouse; and (vi) intercrossing the heterozygote mouse.
[claim3]
3. The Toll-like receptor (TLR)7 homozygous knockout mouse according to claim 2, wherein the targeting vector comprises neomycin resistant gene as a marker and a diphtheria toxin A fragment (DT-A) gene or a herpes simplex virus thymidine kinase (HSV-tK) gene as a negative selection marker in 3'-terminal.
[claim4]
4. The Toll-like receptor (TLR)7 homozygous knockout mouse according to claim 2, wherein whole or a part of Toll-like receptor 7 gene is 1.8-kb TLR7 gene fragment encoding a part of leucine-rich repeat.
[claim5]
5. The Toll-like receptor (TLR)7 homozygous knockout mouse according to claim 4, wherein whole or a part of TLR7 gene is replaced with a neomycin resistant gene.
[claim6]
6. The Toll-like receptor (TLR)7 homozygous knockout mouse according to claim 5, wherein the neomycin resistant gene is pMC1 neo.
[claim7]
7. A method for producing a Toll-like receptor (TLR)7 homozygous knockout mouse whose genome comprises a disruption of a TLR7 gene, wherein the TLR7 homozygous knockout mouse lacks an increase of interferon-alpha compared with a wild-type mouse when R-848 is intraperitoneally injected into the mouse, the method comprising the following steps: (i) constructing a targeting vector by replacing whole or a part of a gene fragment of a gene site including an intracellular region and a transmembrane region of a TLR7 gene obtained by screening a mouse gene library with a plasmid including a poly A signal and a marker gene; (ii) linearizing the targeting vector; (iii) introducing the linearized targeting vector into a mouse embryonic stem cell; (iv) microinjecting the targeted embryonic stem cell whose TLR7 gene function is deficient into a blastocyst of a mouse to generate a chimeric mouse; (v) intercrossing the chimeric mouse with a wild-type mouse to generate a heterozygote mouse, and (vi) intercrossing the heterozygote mouse.
  • Inventor, and Inventor/Applicant
  • AKIRA SHIZUO
  • TOMIZAWA HIDEYUKI
  • YAMAOKA TAKASHI
  • JAPAN SCIENCE AND TECHNOLOGY AGENCY
IPC(International Patent Classification)
Reference ( R and D project ) SORST Selected in Fiscal 2000
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