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III-meeting Muenchen 11 Feb 2006 Improved
Precision
Fiorentina Ascenzioni Dep. Dev. and Cell
Biol Piera Fradiani siRNA selection and
cloning Francesca Tilesi plasmid/siRNA
transfections Valentina Socci lam-siRNA
control Cristina Auriche minichromosome
engineering Rocchi Lucia minichromosome
engineering Irene Bozzoni (Dep. Gent. Mol.
Biol.) psiUx plasmids Alessandro Rosa (Dep. Gent.
Mol. Biol.) psiUx plasmids
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III-meeting Muenchen 11 Feb 2006 Improved
Precision
Tasks within the first 12 month WP1
WP 1 Selection and synthesis of siRNA sequences
and siRNA expressing minichromosomes (F.
Ascenzioni, J. Rosenecker)
available for alpha
Points to discuss
www.improvedprecision.com
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The selected cassettes have been cloned into psiUx
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How to analyse siRNA-mediated downregulation of
ENaC
Plasmid/siRNA transfection reagents
Transient transfectants Stable
transfectants
analysis
Plasmid transfection reagents
selection
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Transient approach
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siRNA efficiency in H441 and A549 cells with anti
laminA/C
About 60 clones/population H441 About 100
clones/population A549
reduction
Lamin A/C immunofluorescence A549/siRNAlam 26
H441/siRNAlam 13 Western blot in progress
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siRNA to down regulate ENaC
pEPIU1-A1/16 pEPIU1- A2/5 pEPIU1- B2/80 pEPIU1-
B3/5 pEPIU1- G1/8 pEPIU1- G2/36
H441 A549
Electroporation
G418 selection
Two population for each transfection with
approximately 50 clones each
RNA extraction (trizol) and semiquantitative
RT-PCR for the alpha. beta and gamma subunits
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RT-PCR of alpha ENaC in H441 and H441A2/A w/wo
dexamethasone induction
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mRNA levels of alpha ENaC in H441 and H441A1/A
normalized for the levels of mRNA of the
endogenous B-actin determined for the same sample
The results refer to three independent experiments
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mRNA levels of alpha ENaC in H441 and H441A2/A
normalized for the levels of mRNA of the
endogenous B-actin determined for the same sample
The results refer to three independent experiments
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III-meeting Muenchen 11 Feb 2006 Improved
Precision
Tasks within the first 12 month WP1
WP 1 Selection and synthesis of siRNA sequences
and siRNA expressing minichromosomes (F.
Ascenzioni, J. Rosenecker)
available for alpha
Points to discuss
www.improvedprecision.com
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Cre-mediated insertion of siRNA cassettes within
minichromosome
loxP
Step 1 Ste2 Step3
loxP
siRNA
Transient expression of Cre
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Starting minichromosomes MC1D are less than 3.5
Mb in size

MC-siRNA
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MC1-D 16/siRNA
MC1-D 16-CAGloxPbsr
37 clones are under investigation to isolate
MC1D-CAGloxPbrs