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Mig-6 Knockout Mice and Elucidation of Association of Mig-6 With Early Onset Degenerative Joint Disease and Role As A Tumor Suppressor

a tumor suppressor and mouse technology, applied in the field of molecular biology and medicine, can solve the problems of affecting the quality of life, acute and chronic pain and disability, and astronomical costs of pain relief medications, so as to delay development, delay the effect of development, and evaluating the appearance, growth or metastasis rate, growth rate, and the time of appearan

Inactive Publication Date: 2013-09-12
VAN ANDEL RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The patent describes the discovery of a gene called MiG6, which is commonly mutated in lung cancer and other cancers. When MiG6 is deleted in mice, they develop lung and gallbladder tumors. The patent also describes a method for creating a knockout mouse with a disrupted MiG6 gene, which results in enhanced tumor formation and joint abnormalities that resemble osteoarthritis. These animals can be used as a model for studying the role of MiG6 in cancer development and for developing new treatments. Overall, the patent presents a valuable resource for research on cancer biology and therapeutic development.

Problems solved by technology

J. Rheumatol. 16:427-41, 1989), and greatly interferes with quality of life by causing acute and chronic pain and disability.
In addition, the costs for pain relief medication are astronomical.
Although several genetic and biomechanical factors including heredity, obesity, injury and joint overuse are thought to contribute to the development of osteoarthritis, the molecular mechanism underlying this disease is still elusive.
Indeed, no mutations have been reported in Mig-6 to date, and the role of Mig-6 in human lung, gallbladder, and bile duct carcinogenesis has not been assessed.

Method used

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  • Mig-6 Knockout Mice and Elucidation of Association of Mig-6 With Early Onset Degenerative Joint Disease and Role As A Tumor Suppressor
  • Mig-6 Knockout Mice and Elucidation of Association of Mig-6 With Early Onset Degenerative Joint Disease and Role As A Tumor Suppressor
  • Mig-6 Knockout Mice and Elucidation of Association of Mig-6 With Early Onset Degenerative Joint Disease and Role As A Tumor Suppressor

Examples

Experimental program
Comparison scheme
Effect test

example i

Mig-6 Knockout Mice: Materials and Methods

[0112]Mice and Genotyping.

[0113]To generate Mig-6 knockout mice, the present inventors constructed a Mig-6 targeting vector or knockout vector (FIG. 1A) by inserting a 5 kb genomic fragment upstream of exon 2 (designated e2) and a 3 kb genomic fragment downstream of exon 4 (designated e4) into the pPNT vector (Tybulewicz, V et al., (1991) Cell 65:1153-63), respectively 5′ to and 3′ to the PGK-NEO cassette.

[0114]As shown in FIG. 1A, this knockout vector or “targeting vector” includes the following components (in the 5′-3′ direction):[0115](1) The 5′-homologous recombination sequence derived from mouse Mig-6 genomic DNA; this is a ˜5-kb genomic fragment mostly upstream of exon 2 (though it includes a short sequence from exon 2). This fragment has the following sequence [SEQ ID NO:19]. The bold / underscored 3′ sequence corresponds to nt's from exon 2.

AGGTCATCTA GTGGAGGCAA GAACACACAA ACCATCCTTT CTTTGCATCC TTTTGGACAGCATTTATGAA ATATTTGCTG AAGCTATCA...

example ii

Early Onset Osteoarthritis in Mig-6 Knockout Mice

[0128]Mig-6 deficient mice were generated by conventional gene targeting technology by replacing the entire coding region of Mig-6 with PGK-Neo cassette / construct (FIG. 1A). The loss of wild type alleles of Mig-6 was determined by Southern Blot analysis using “Probe A” and by PCR-based genotyping (FIG. 1B).

[0129]Probe A has the following nucleotide sequence [SEQ ID NO:21]:

TACCTGCCTT ATTCAGAGGA GTCAAGTGTG TATCTTAAGT CATTTTGTTC CAGTAATTTGAAGAGCCTAA GACTTTAAAA GAGAGGCTGT GGTATGGTCG AGAGCATAAA CTTTGAGGCCAAGCTTCCTG AAGTAAGCCG TGGCATTACT GTGGCTCACC GGAGCCGAGT CAGATCTAGTTGCAGAAGCT CCTCGTCTGT CATTGAGAGT AGTGTCCCAC CTACCTTAGG GCTGCTACAAGGATAAAACT GAAAACCTTC CTGACAGACA GTATCCTATG AATGTCATTA TCATCACCTATGTATTAATT TTAACTCTCC TGAGTTGTCC ATTGGGTTAT TTAAATGCTT GTTAAATAAACTTGAAGTTT TAAAGACTCA TTTCCCATCA TTAGCCCATT GTGGTCATTG TCATTAAGATTACAACAGAA TCCACACATC GTTCACAGGT ACAGTGCATT GCATATGTCG GAAAGAAATGCTCTTCCATG CCGTGTGTGC TTGCCTGTGT CTGTGGATGG TACTGTTGA...

example iii

Mig-6 and Tumor Suppressor Gene Activity: Experimental Procedures

Human Lung Cancer Cell Lines and Cell Culture

[0142]The nine non-small cell lung cancer (NSCLC) cell lines EKVX, HOP62, HOP92, NCI-H23, NCI-H226, NCI-H322M, NCI-H446, NCI-H522, and A549 were derived from the NCI 60 cell lines. The cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum.

Mutational Analysis of Mig-6

[0143]Human lung cancer and normal control tissue was obtained through the Cooperative Human Tissue Network (CHTN). Genomic DNA was isolated from human cell lines and tissues. Polymerase chain reaction (PCR) was performed to amplify the entire coding regions of Mig-6 (exons 2, 3, and partial exon 4) using three primer pairs. Primers used for screening genomic DNA mutations in the coding region of human Mig-6 are as follows. The same set of primers is also used for sequencing PCR products for determining if there is a mutation or not in the coding region of human Mig-6.

(1) Pair-I for amp...

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Abstract

The molecular mechanism underlying degenerative joint disease, also known as osteoarthritis (OA), is not fully understood. Disruption of mitogen inducible gene 6 (Mig-6) in mice by homologous recombination (KO mice) led to early onset OA as revealed by simultaneous enlargement and deformity of multiple joints, degradation of articular cartilage and the development of bony outgrowths or osteophytes within the joint space. The latter appeared to be derived from proliferation of mesenchymal progenitor cells followed by differentiation into chondrocytes. Because of the striking similarity to human OA, Mig-6 KO mice arc a useful animal model for studying the mechanism of this disease and for testing new drugs or therapies for treating OA. These KO mice also developed epithelial hyperplasia, adenoma, and adenocarcinoma in organs such as lung, gallbladder, and bile duct. Mig-6 is therefore a tumor suppressor gene and is a candidate gene for the frequent Ip36 genetic alterations found in lung cancer. It can be used as a tumor biomarker as well as a target for cancer therapy. Mig-6 is located in human chromosome Ip36, a locus frequently associated with human lung cancer. Mig-6 is a negative regulator of EGF signaling, and like EGF, was induced by HGF / SF in human lung cancer cell lines. Frequently the receptors EGFR and Met were co-expressed, and Mig-6 was induced by both EGF and HGF / SF in a MAPK-dependent fashion. Not all tumor lines express Mig-6 in response to either EGF or HGF / SF. In these cases, missense and nonsense mutations in the Mig-6 coding region were found, as was evidence for Mig-6 transcriptional silencing.

Description

CROSS REFERENCE TO RELATED APPLICATION[0001]This application is a divisional of U.S. application Ser. No. 11 / 917,557 filed Jun. 15, 2006, which is the U.S. National Phase of PCT Application Serial No. PCT / US2006 / 023257, filed Jun. 15, 2006, which claims the priority of U.S. Provisional Application Ser. No. 60 / 690,493, filed Jun. 15, 2005 and U.S. Provisional Application Ser. No. 60 / 789,612 filed Apr. 6, 2006; each of which is incorporated herein by reference in its entirety.BACKGROUND OF THE INVENTION[0002]1. Field of the Invention[0003]This invention in the field of molecular biology and medicine is directed to knockout mice in which the mig-6 gene is disrupted resulting in animals which develop early-onset joint abnormalities characteristic of osteoarthritis and are highly susceptible to tumorigenesis of a number of types of cancer, primarily lung cancer. These animals serve as models for testing potential drugs and other therapeutic measures to prevent or treat osteoarthritis, an...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12Q1/68
CPCA01K67/0276A01K2217/075A01K2227/105A01K2267/03C12Q1/686C12N15/8509C12N2517/02C12N2800/30A01K2267/0368
Inventor ZHANG, YU-WENVANDEWOUDE, GEORGE F.
Owner VAN ANDEL RES INST
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