Nobel Lecture Gene Targeting into the 21 st Century Mario R. - - PowerPoint PPT Presentation

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Nobel Lecture Gene Targeting into the 21 st Century Mario R. Capecchi Howard Hughes Medical Institute Department of Human Genetics University of Utah School of Medicine December 7 th , 2007 How did the idea to do Gene Targeting arise?

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Nobel Lecture

Gene Targeting into the 21st Century

Mario R. Capecchi Howard Hughes Medical Institute Department of Human Genetics University of Utah School of Medicine December 7th, 2007

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How did the idea to do Gene Targeting arise?

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Considerations for Models

1. Inducing Event
2. Time of Induction
3. Stochiometry
4. Micro-molecular

and -cellular environment

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PAX3-FKHR Structure

Paired Box DBD Octapeptide DBD Homeodomain Partial Transactivation Domain Transactivation Domain 3’ UTR Ser 253 Partial Forkhead Domain

Barr et al, 1993

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Modeling Alveolar Rhabdomyosarcoma

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What’s Important?

Pax3:Fkhr Fkhr:Pax3

Pax3:Fkhr gene is created. Fkhr:Pax3 is created. Pax3 allele is destroyed. Fkhr allele is destroyed.

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Chromosomal Translocations: Why not generate real translocations?

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Pax3 becomes Pax3:Fkhr

with Time- & Tissue Specificity

3’ Fkhr IRES-eYfp Stop! Pax3 Locus 3’ Fkhr IRES-eYfp

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Conditional Knock-In Construct

Pax3 Ex 8 - 10 Pax3 Ex 7 LoxP pA LoxP Fkhr In 2 - Ex 3 Fkhr 3’UTR (6.4 kB) IRES

eYfp

Pax3 3’UTR TK1 FRT-Neo-FRT WT P3F Germline Offspring P3F/WT

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Pax3 WT / P3Fm

Pax3 LacZ / wt Pax3 (E10.75)

Mansouri et al, 1997

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FKHR Oxidative Stress DNA Damage Differentiation Growth Arrest

r Apoptosis

Reversal of Oxidation

r DNA Damage

G1 or M Cell Cycle Arrest Akt PTEN

DYRK1A RAS/RAL Crm-1

FKHR Integrates Repair Signals

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RMS Cells are not from a Distinct Embryonic Subset

Differentiated Myofiber (Myf6, MHC, MCK, SMA, Desmin, Vimentin) Myoblast (MyoD, Myf5) Myogenic Progenitor (Pax3, Pax7, c-Met/HGF, Msx1, Lbx1)

Goulding et al, 1994 Williams & Ordahl, 1994 Zhang et al, 1995 Gross et al, 2000 Dietrich et al, 1999 Hasty et al, 1993 Kablar et al, 1997 Schafer et al, 1994

Myotube (Primary, Secondary) (Myogenin, Desmin)

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RMS Cells are not from a Distinct Postembryonic Subset

Seale et al, 2000 Odelberg et al, 2000 (Side Population Cell)

Msx1

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Pax3 becomes Pax3:Fkhr

with Time- & Tissue Specificity

3’ Fkhr IRES-eYfp Stop! Pax3 Locus 3’ Fkhr IRES-eYfp

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Pax3:Fkhr is expressed in the Pax3 domain

Pax3 P3Fa / wt eYFP (E8.75) Pax3 P3Fa / wt eYFP (E10.5)

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Pax3 P3Fm / wt Pax3 P3Fa / wt

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RMS Cells are not from a Distinct Postembryonic Subset

Seale et al, 2000 Odelberg et al, 2000 (Side Population Cell)

Msx1

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Hardwire Pax7-Cre

4776-N1

Pax7-IRES-Cre

Pax7 IRES Cre frt Stop!

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10.5 g 3 g

Pax3 P3Fm / wt Pax3 P3Fa / wt

activated by

Pax7 ICNm / wt

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Satellite Cells are Present, but Reduced?

Pax3 P3Fa / wt EM (P0)

SC MN

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Despite the fusion gene Pax3:Fkhr being expressed in every satellite cell during and post fetal development these mice did not develop Alveolar Rhabdomyosarcomas.

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Temporal, Muscle-Specific Conditional Cre

Myf6 IRES Stop! Tet-On TRE + Dox PminCMV Cre rtTA tCre/WT

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WT (background) P3F x tCre (triggered)

eYFP Marker is Induced, Heterogeneously

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MUSA

U286 (12 months

ARMS in Pax3 P3Fm/WT Myf6 tCreM/WT mice

Myogenin

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ARMS in Pax3 P3Fm/P3Fm Myf6 ICNm/wt Trp53 F2-10/wt mice

U4387 (6 months

Myogenin

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Developing a Mouse Model of Synovial Sarcoma