Kevin B. Jones, MD Charles Searby, BS Gail Kurriger, BS James Martin, PhD Peter J. Roughley, PhD Jose A. Morcuende, MD, PhD Joseph A. Buckwalter, MD, MS Val C. Sheffield, MD, PhD Department of Orthopaedics and Rehabilitation University of Iowa Hospitals & Clinics Osteochondromagenesis: Loss of heterozygosity modeled via Cre- mediated inversion of the second exon of Ext1 in chondrocytes Connective Tissue Oncology Society London, UK Friday, 14 November 2008
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Kevin B. Jones, MD Charles Searby, BS Gail Kurriger, BS James Martin, PhD Peter J. Roughley, PhD
Osteochondromagenesis: Loss of heterozygosity modeled via Cre-mediated inversion of the second exon of Ext1 in chondrocytes. Department of Orthopaedics and Rehabilitation University of Iowa Hospitals & Clinics. Kevin B. Jones, MD Charles Searby, BS Gail Kurriger, BS James Martin, PhD - PowerPoint PPT Presentation
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Kevin B. Jones, MDCharles Searby, BSGail Kurriger, BS
James Martin, PhDPeter J. Roughley, PhD
Jose A. Morcuende, MD, PhDJoseph A. Buckwalter, MD, MS
Val C. Sheffield, MD, PhD
Department ofOrthopaedics and RehabilitationUniversity of IowaHospitals & Clinics
Osteochondromagenesis: Loss of heterozygosity modeled via Cre-mediated inversion
• Loss of heterozygosity in chondrocytes of cartilage cap of HME osteochondromas
• Both alleles somatically mutated in some solitary osteochondromas
Raskind et al. 1995; Bovee et al. 1999; Hecht et al. 1995 and 1997.
Ext1 Knock-Out Mice by Lin et al. 2000
Mouse Ext1 has 99% Homology to Human EXT1
PROBLEMS:• Homozygous Ext1-knock-out mice do not
survive to birth• Heterozygous Ext1-knock-out mice very
rarely form osteochondromas
Simlar with Ext2 knock-out mice, Sitckens et al. 2005.
Of Mice and Men
Of Mice and Men
~60 kg Human flesh
~30gMouse flesh
~2000X the number of cells
Haploinsufficiency
or
Loss of Heterozygosity?
Methods
CRE
loxP loxPLoxP LoxP
Cre
Routine or cis orientation of loxP sites results in fragment excision
trans orientation of loxP sites results in reversible fragment inversion
CRE
Pxol loxP
CODING SEQUENCE
ECNEUQES GNIDOC
LoxP PxoL
Cre
CODING SEQUENCE
ECNEUQES GNIDOC
You do the math. . .
• Cre-mediated inversion yields 40% reverse orientation per trans-floxed allele
• Homozygosity for trans-floxed alleles should yield 40% loss of total copies of functional gene across tissue– 20% of cells will remain unaffected and fwd/fwd
– 20% of cells will end up fwd/fwd after inversion
– 40% of cells will end up fwd/rev after inversion
– 20% of cells will end up rev/rev after inversion