Conditional dicer gene depletion in the postnatal myocardium

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Conditional Dicer Gene
Depletion in the
Postnatal Myocardium
Provokes Spontaneous
Cardiac Remodeling
MARTINS, P. A. C.; BOURAJJAJ, M.; GLADKA, M.;
KORTLAND, M.; OORT,R. J. V.; PINTO, Y. M.;MOLKENTIN, J. D.; WINDT, L. J.

Circulation, v. 118, n. 15, oct. 2008
American Heart Association

Aluna: Dayana P. P. Siqueira

INTRODUCTION
miRNA
Endogenous
Regulate gene
expressionSmall
noncoding

Drosha
Dicer

Mature miRNA
Pri-miRNA

(18 to 24 nucleotides long)

AMBROS, 2003; FARH, et al., 2005; PASQUINELLI, HUNTER, BRACHT, 2005; CULLEN, 2004.

RNA-induced
silencingcomplex

BARTEL, 2004; BENTWICH et al., 2005; BEREZIKOV et al., 2005.

Negatively regulate
protein expression by
either mRNA
degradation or
translational inhibition

miRNA

May regulateup 1/3 of the
human genome

Current studies

Multiple transcripts

Fundamental role in
regulating gene
expression

OBJECTIVES

Trigger loss of Dicer in the postnatal myocardiumthrough the use of a tamoxifen-inducible Cre
recombinase expressed in murine heart cells.

METHODS
Transgenic Mice

Cre

Mice were treated with:
• vehicle or tamoxifen (20mg/Kg per day)
•intraperitoneal injections for consecutive 5 days

3 weeks

8 weeks

Western Blotting
Proteins were extracted from mouse cardiac tissue

Antibodies against:

Dicer (rabbit polyclonal)
GAPDH(mouse monoclonal)

Irx5 (mouse monoclonal)
Kcnd2 (rabbit polyclonal)

Histological Analysis and Immunofluorescence Microscopy

Hearts were arrested in diastole
Perfusion-fixed with 4%paraformaldehyde
Embedded in parafin

Sectioned at 5µm
H&E

routine histological analysis

Sirius red

detection of fibrillar collagen

WGA

visualize and quantify the cross-sectional area ofcardiomyocytes

CD31

visualize and quantify the number of capillaries per
myofiber

Transthoracic Echocardiography

Were performed on mice anesthetized with isoflurane
1 week and 4 weeks...
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