PowerPointPrsentation

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Systematic Production of Multipurpose Alelles in
Mouse Embryonic Stem Cells
Harald von Melchner University of
Frankfurt GERMANY
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Objectives

• 12,000 conditional mutations by gene trapping-
  5,000 conditional mutations by targeted
  trapping- 3,000 conditional mutations by gene
  targeting

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Typical gene trap cassette
SA
Reporter
pA
SD
SA
pA
pA
SA
Reporter
E1
E2
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Gene traps simultaneously

• (i) mutate
• (ii) tag
• (iii) report

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Directional recombination using heterotypic RTs
(FlEx)

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Gene inactivation by a conditional cassette
.
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Possible limitations of conditional gene trapping
Minus strand insertions of large open reading
frames could be mutagenic !!
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ES cell lines used for gene trap validation
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Conditional inactivation of RBBP7
RT-PCR
X-Gal
Immunoblot
FLPe
Cre
FLPe
Cre
wt
t
inv
re-inv
wt
t
inv
re-inv
endo
fus
endo
fus
endo
fus
endo
fus
RBBP7
lamin A
wt
t
inv
re-inv
??? pol II
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Conditional inactivation of ATRX
RT-PCR
Immunoblot
SA
?geo
pA
E1
E2
E8
wt
t
inv
re-inv
wt
t
inv
re-inv
endo
fus
endo
fus
endo
fus
endo
fus
ATRX
lamin A
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Conditional inactivation of Glt28d1
RT-PCR
Northern blot
E7
E11
E10
E13
pA
Ceo
Cre
FLPe
Cre
FLPe
wt
t
wt
t
inv
re-inv
inv
re-inv
endo
fus
endo
fus
endo
fus
endo
fus
Glt28d1
fus
wt
t
inv
re-inv
GAPDH
GAPDH
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München
Frankfurt
Berlin
Johann Wolfgang Goethe University Medical School
Max Planck Institute for Molecular Genetics
GSF Research Center for Environment and Health
clone production
vector design
clone production distribution
RACE and sequencing
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GGTCs conditional gene trap resource
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www.genetrap.org
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Conditional Gene Trap Cassettes

• Induce germline and somatic mutations in the
  mouse
• Enable analysis of gene function in a temporally
  and
• spatially controlled manner
• Create multipurpose alleles amenable to a variety
• of postinsertional modifications by recombinase
  mediated
• cassette exchange (RMCE)
• Can be used for targeted trapping and gene
  targeting

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Frank Schnütgen Julia Schmidt
Petra Van Sloun
Silke Garkisch
Corinna Strolz
Silke De-Zolt
Joachim Altschmied
BERLIN GROUP Patricia Ruiz Carsta
Werner Carsten Horn
MUNICH GROUP Wolfgang Wurst Melanie
Hollatz Thomas Floss Jens Hansen