Methods

mouse_in_handIn our research we use conditional knockout and transgenic mice as well as isolated cardiomyocytes

All experimental work planned and provided accordingly DIRECTIVE 2004/9/EC OF THE EUROPEAN PARLIAMENT AND OF THE COUNCIL of 11 February 2004 on the inspection and verification of good laboratory practice (GLP). All experimental procedures with animals approved by the IMBG Animal Care and IMBG Bioethics Committee.

aMHC–Cre mice »

aMHC–Cre mice express the bacterial Cre-recombinase controlled by the promoter of the α-myosin heavy chain (αMHC or alpha-MHC; Myh6) , It is important to note that in our model Cre-recombinase was active under the control of a tissuespecific promoter exclusively in cardiomyocytes, starting approximately on the sixth day of embryonic development, i.e., after the lining of the embryonic heart. Mice were generate by Dr.Michael Schneider (Baylor College of Medicine, United States) and kindly provided by Dr. G. L. Radice (Jefferson Medical College, USA).

Inducible aMHC–Cre mice »

Inducible aMHC–Cre mice or alpha-MHC-MerCreMer (αMHC-MerCreMer) transgene has the mouse cardiac-specific alpha-myosin heavy chain promoter (αMHC or alpha-MHC; Myh6) directing expression of a tamoxifen-inducible Cre recombinase (MerCreMer) to juvenile and adult cardiac myocytes. These αMHC-MerCreMer transgenic mice allow the creation of bitransgenic mice for Cre-lox studies of temporally regulated deletion of loxP-flanked targeted genes in cardiac tissues/cells. Mise were created by Philippe Soriano and obtained from Jackson Laboratories (Bar Harbor, ME, USA) and kindly provided by Dr. G. L. Radice (Jefferson Medical College, USA).

R26R reporter mice (or B6.129S4-Gt(ROSA)26Sortm1Sor/J) »

R26R reporter mice (or B6.129S4-Gt(ROSA)26Sortm1Sor/J) may be used as a Cre-reporter strain; to test the tissue/cellular expression pattern of cre transgenic mice. They expressed the bacterial beta-galactosidase (lacZ) when crossed to a Cre recombinase-expressing strain, lacZ expression is observed in the cre-expressing tissues. Mise were created by Philippe Soriano and obtained from Jackson Laboratories (Bar Harbor, ME, USA) and kindly provided by Dr. G. L. Radice (Jefferson Medical College, USA).

BAT-GAL transgenic mice »

BAT-GAL transgenic mice (also called β-catenin/TCF/LEF reporter transgenic mice) are a reporter strain that expresses beta-galactosidase in the presence of activated beta-catenin (mimics the pattern of Wnt signaling). These mice may be useful for identifying mutations that affect the Wnt-signalling pathway, and in the identification of Wnt-responsive cell populations in development and disease. kindly provided by Dr Mathew Weller and Prof Thomas Brown (Max-Planck-Institute for Heart and Lung Research, Bad Nauheim, Germany)

N-cad flox/flox »

N-cad flox/flox  mutant mice possess loxP sites flanking exon 1 of the Cdh2(or Ncad) gene. This strain was generated by Dr Igor Kostestkii and Dr. Glenn Radice and kindly provided by Dr. G. L. Radice (Jefferson Medical College, USA).

α-E-catenin flox/flox mice »

α-E-catenin flox/flox mice These floxed mutant mice possess loxP sites flanking exon 2 of the Ctnna1 (α-E-catenin ) gene. Mice were obtained from Jackson Laboratories (Bar Harbor, ME, USA) and kindly kindly provided by Dr. G. L. Radice (Jefferson Medical College, USA).

β-catenin flox/flox mice »

β-catenin flox/flox mice possess loxP sites located in introns 1 and 6 of the targeted gene. Mice were generated with C57BL/6J blastocysts  and wild-type C57BL/6J mice using. This mice were obtained from Jackson Laboratories (Bar Harbor, ME, USA) and kindly provided by Dr. G. L. Radice (Jefferson Medical College, USA).

Wif1 flox/flox mice »

Wif1 flox/flox  mice possess loxP sites located in some introns of targeted gene. The protein encoded by this gene functions to inhibit WNT proteins, which are extracellular signaling molecules that play a role in embryonic development. This protein contains a WNT inhibitory factor (WIF) domain and five epidermal growth factor (EGF)-like domains. Mice were generated and kindly provided by Dr Mathew Weller and Prof Thomas Brown (Max-Planck-Institute for Heart and Lung Research, Bad Nauheim, Germany)

 

To obtain a cardiac specific ablation of the target gene, we crossed mice that express the bacterial Cre-recombinase controlled by the promoter of the α-myosin heavy chain ((MHC)–Cre) and that are heterozygous for the conditional knockout β-catenin ((αMHC)–Cre; β-catflox/wt) and the animals homozygous for the conditional knockout β-catenin (β-catflox/flox).

Extending training for hypertrophy inducing

The 3-months-old male animals both Mut and Cont were used for the swimming test (physiological hypertrophy modeling experiments). The protocol for the swimming test was adopted from ( Evangelista FS, Brum PC, Krieger JE. 2003).

Isolated cardiomyocytes

 

Methods: cells isolation and cultivation, mice manipulation, IV, IP and IM injections, mice operations; MTT-test; HE-staining, MT-staining, vanGisen staining, Immunohistochemistry; Paraffin embed section preparing; protein isolation and Western-blot; PCR; qPCR; RNA and DNA – isolation; ChIP-PCR an ChIP-seq. Statistics: one- and two-way ANOVA test; Coefficient of variation; Student test; Bioinformatics analysis