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

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?

Considerations for Models 1. Inducing Event 2. Time of Induction 3. Stochiometry 4. Micro-molecular and -cellular environment

PAX3-FKHR Structure Ser 253 Partial Paired Box Homeodomain Forkhead 3’ UTR DBD Domain Partial Octapeptide Transactivation Transactivation DBD Domain Domain Barr et al , 1993

Modeling Alveolar Rhabdomyosarcoma

What’s Important? Pax3:Fkhr Fkhr:Pax3 Pax3:Fkhr gene is created. Fkhr:Pax3 is created. Pax3 allele is destroyed. Fkhr allele is destroyed.

Chromosomal Translocations: Why not generate real translocations?

Pax3 becomes Pax3:Fkhr with Time- & Tissue Specificity Stop! 3’ Fkhr 3’ Fkhr Pax3 Locus IRES-eYfp IRES-eYfp

Conditional Knock-In Construct Pax3 Pax3 Fkhr Pax3 Fkhr IRES pA 3’UTR TK1 Ex 7 Ex 8 - 10 In 2 - Ex 3 3’UTR - eYfp (6.4 kB) LoxP LoxP FRT- Neo -FRT Germline Offspring P3F/WT WT P3F

Pax3 LacZ / wt Pax3 (E10.75) Pax3 WT / P3Fm Mansouri et al , 1997

FKHR Integrates Repair Signals PTEN Differentiation Akt Oxidative Stress Growth Arrest or Apoptosis FKHR Reversal of Oxidation or DNA Damage DNA Damage DYRK1A RAS/RAL Crm-1 G 1 or M Cell Cycle Arrest

RMS Cells are not from a Distinct Embryonic Subset Myogenic Progenitor (Pax3, Pax7, c-Met/HGF, Msx1, Lbx1) Myoblast (MyoD, Myf5) Myotube (Primary, Secondary) (Myogenin, Desmin) Goulding et al , 1994 Williams & Ordahl, 1994 Zhang et al , 1995 Differentiated Myofiber Gross et al , 2000 (Myf6, MHC, MCK, SMA, Dietrich et al , 1999 Desmin, Vimentin) Hasty et al , 1993 Kablar et al, 1997 Schafer et al, 1994

RMS Cells are not from a Distinct Postembryonic Subset (Side Population Cell) Msx1 Seale et al , 2000 Odelberg et al , 2000

Pax3 becomes Pax3:Fkhr with Time- & Tissue Specificity Stop! 3’ Fkhr 3’ Fkhr Pax3 Locus IRES-eYfp IRES-eYfp

Pax3:Fkhr is expressed in the Pax3 domain Pax3 P3Fa / wt Pax3 P3Fa / wt eYFP (E10.5) eYFP (E8.75)

Pax3 P3Fm / wt Pax3 P3Fa / wt

RMS Cells are not from a Distinct Postembryonic Subset (Side Population Cell) Msx1 Seale et al , 2000 Odelberg et al , 2000

Hardwire Pax7-Cre frt Pax7 IRES Cre Stop! Pax7-IRES-Cre 4776-N1

10.5 g 3 g Pax3 P3Fa / wt Pax3 P3Fm / wt activated by Pax7 ICNm / wt

Satellite Cells are Present, but Reduced? Pax3 P3Fa / wt EM (P0) SC MN

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

Temporal, Muscle-Specific Conditional Cre + Dox Tet-On TRE Myf6 rtTA Stop! Cre IRES P minCMV tCre/WT

eYFP Marker is Induced, Heterogeneously WT (background) P3F x tCre (triggered)

ARMS in Pax3 P3Fm/WT Myf6 tCreM/WT mice U286 (12 months old) Myogenin MUSA

ARMS in Pax3 P3Fm/P3Fm Myf6 ICNm/wt Trp53 F2-10/wt mice U4387 (6 months old) Myogenin

Developing a Mouse Model of Synovial Sarcoma