A Flexible Toolkit

1
FLPFLEX
A Flexible Toolkit for Controlling Gene
Expression in the Mouse SPECIFIC TARGETED
RESEARCH OR INNOVATION PROJECT Thematic Priority
1.1.1 Life sciences, genomics and biotechnology
for health Proposal/Contract no. 513769
Grant duration 7/2005 - 6/2008
2
FLPFLEX Partners
Nadia Rosenthal EMBL Andrea Ballabio TIGEM,
Italy Wolfgang Wurst GSF, Germany Riccardo
Cortese IRBM, Italy Hansjörg Hauser GBF, Germany

3
FLPFLEX Rationale

• Mammalian system complexity
• Many cell types function in each tissue
• Many tissue types function in each organ
• Many organs function in each system
• Many stage-specific functions in each disease
• Our control of mouse gene expression in space and
  time needs better resolution for modelling human
  disease

4
FLPFLEX Fulfilling the need for higher
resolution
To meet the increasing need for tissue-specific
and/or regulated gene expression for multiple
applications in the mouse.
5
FLPFLEX Mission

• FLPFLEX is dedicated to the development of novel
  tools to manipulate gene expression
• for dissecting complex genetic pathways
• for generating more accurate models of
• human disease.

6
FLPFLEX Approach

• FLPFLEX cassette exchange allows multiple rapid
  manipulations in a single genomic locus
• by engaging the cells own transcriptional
  regulatory circuits to capture novel expression
  patterns
• by providing powerful and adaptable tools for
  designing increasingly complex genetic models of
  human physiology and pathology

7
Conventional gene trapping
ATG
P
x
ex1
ex2
ex3
pA
Endogenous locus
?geo Vector
Trapped allele
ATG
P
ex1
ex2
ex3
pA
No further modification possible
8
Limitation of conventional gene trapping
P
ex1
ex2
ex3
pA
Interesting trapped locus
P
LacZ
SA
Neo
pA
pA
ex1
ex2
ex3
P
ex3
pA
transactivator
P
toxin
ex3
pA
P
ex3
pA
Cre
P
Gene of interest
ex3
pA
For each construct, an additional homologous
recombination must be performed in the trapped
locus
9
FLPFLEX flexible gene switching
FLPFLEX allele
ATG
P
Reporter
SA
Selector
pA
ex1
ex2
pA
ex3
Gene of interest - Cre - transactivator -
toxin - knock-in
F
One trapped locus, multiple gene readouts with
rapid enzymatic recombination
10
FLPFLEX tools
Aim
Tool


11
A. Generation of FLPFLEX ES cell clones
SA
ATG
LacZ
SA
Neo
pA
FlipRosaßgeo (EUCOMM)
SA
ES cells vector
Electroporation
Selection in G418 picking
Growing colonies to confluency
Storage of master plate
bgal activity
12
B. Selection of FLPFLEX ES cell clones
?gal positive FLPFLEX clones
FISH analysis
mRNA Extraction
5 RACE
DNA sequencing, in silico analysis
Automated ISH of adult mouse tissues
Selection of expression Pattern of interest
13
C. Generation of FLPFLEX mice
Injection of ?gal-positive FLPFLEX ES cell into
mouse blastocyst
Chimera production Germline transmission Analysis
of ?gal expression patterns
14
FLPFLEX proof of concept GFP cassette exchange
Trapped gene expression GFP reporter
expression
In situ hybridisation
GFP is flipped into in FLFPEX targeted locus in
ES clone GFP reporter accurately reflects trapped
gene expression pattern Trapped gene regulation
is not perturbed by cassette exchange
15
FLPFLEX Key features

• Multifunctional readout vectors
• A library of expression patterns
• C) Automated high-throughput screening for clones
  
• D) Automated high throughput expression analysis
• E) Flexible disease modeling in mice
• F) FLPFLEX public database (with EUCOMM)
• G) Dissemination of material

16
FLPFLEX A complement to EUCOMM

• Harmonised vectors
• Complementary set of targeted genes
• C) Emphasis on useful expression patterns
• D) Rapid proof of concept
• E) Pipeline to Cre Zoo

17
FLPFLEX