Worth noting, mouse sik1 maps on mouse chromosome 17 in a region that is syntenic with human chromosome 21q22.3, where the human ortholog is located. It is well known that trisomy of 21 leads to Down syndrome, the most common genetic cause of mental retardation in the human population. Notably, forty percent of newborns with Down syndrome have congenital heart defects (CHD), of which the most frequent are atrioventricular septal defects [31]. Molecular studies of rare individuals with CHDs and partial duplications of chromosome 21 have estab- lished a candidate region of 5 Mb in which SIK is localized [32]. Given that sik1 is specifically involved in cardiomyogenesis, we can speculate that this gene can play a role in CHDs observed in Down syndrome patients, opening new avenues in the under- standing of the biological pathways involved in this severe disorder. Materials and Methods Ethics Statement The experiments with murine cell lines were performed in compliance with the institutional guidelines (Italian Legislative Decree Nu 120/92 (published on February 18th 1992) being the official Italian publication of the European Directive Nu 88/320/ CEE and 90/18/CEE concerning matters of inspections and verifications of Good Laboratory Practices). The parental cell line E14Tg2a.4 was obtained by Baygenomics, previously described in Brennan J, Skarnes WC. [33]. The generation of sik1wt/flp cell line was described in Cobellis, G et al. [14] and the sik1flp/flp and HAsik1 cell lines were obtained by further modifications of sik1+/flp cell line described in this report Plasmids rSik1 coding was obtained digesting the plasmid pGFPC-rSik1 BamHI/EcoRI and cloned into the expression vector pcDNA3X(+)HA (Sigma-Aldrich). The sik1 fragment encoding the amino acids 1–249 was amplified from ES cells cDNA with the primers sik1HAfor 59-CTCGAGATGGTGATCATGTCGGAGT-39 and sik1HArev 59-TCTAGATCACTAAGGGTTCTTCTTCTTTGG- T-39 containing the XhoI and XbaI sites, respectively. The PCR product was subcloned in pCR-Blunt II-TOPO vector (Invitrogen), digested XhoI/XbaI from the TOPO Blunt II and cloned into pcDNA3X(+)HA. The plasmid pGFPC-rSik1 was a kind gift of Dr. Hiroshi Takemori. Cell Culture The feeder-independent mouse ES cell lines E14 Tg2A.4 (wild- type, sik1wt/flp, sik1flp/flp and sik1flp/flp + HAsik1) were grown in DMEM supplemented with 15% (vol vol) fetal bovin serum, 2 mM L-glutamine, 1 mM MEM sodium pyruvate, 0.1 mM 2- mercaptoethanol (all from Invitrogen), 1,000 units/ml leukemia inhibitory factor (Esgro, Chemicon). HEK293T cells were maintained in DMEM (Invitrogen) containing 10% of fetal bovine serum (Hyclone). Production and Identification of Homozygous Mutant ES Cells In order to obtain homozygous sik1flp/flp ES cells, heterozygous sik1wt/flp ES cells were cultured in increasing concentrations of G418 (2 and 3 mg/ml) for 14 days as previously described [15]. After the selection, to identify homozygous sik1flp/flp ES cells, 96 surviving clones were analyzed by RT-PCR using the following primers: sik1UP (59-TCGGTGTGGTGCTGTACGT-39), sik1LW (59-AC- Sik1 Controls Cardiomyogenesis TGCTGGGGGAGATGGAT-39) and L232 (59-GATGTGCTG- CAAGGCGATTA-39), as depicted in figure 1. Production of sik1flp/flp + HAsik1 and p57 ES Cells The sik1flp/flp ES cell line was electroporated with 4 mg plasmid vector Pallino b-actin using the Nucleofector II (Amaxa biosys- tem). This plasmid vector contains the HAsik1 cDNA, driven by the b-actin promoter, and the puromycin resistance gene, driven by the phospho-glycerokinase promoter. The plasmid used to introduce p57 contains the p57 cDNA driven by PGK promoter. The stable selection marker is puromycin. In order to identify HAsik1 or p57 overexpressing ES cells, two weeks after selection with 1.2 mg/mL puromycin, the resistant colonies were screened by RT-PCR using the primers sik1UP and sik1LW for HAsik1 clones and with vector up and p57low for the p57 clones. ES Cells Differentiation ES cells were differentiated by EB formation using the ‘‘hanging drop’’ method as previously described [13,34]. The cardiomyocyte differentiation medium consists of the ES cells medium depleted of LIF. The neuronal and glial differentiation medium consists of the Knockout medium (Invitrogen) supplemented with 15% Knockout Serum SR, 2 mM L-glutamine, 1 mM sodium pyruvate, 0.1 mM 2-mercaptoethanol (all from Invitrogen) and depleted of LIF. Cell Proliferation Assay Wild type and sik1flp/flp ES cells (56104 cells) were subjected to a pulse with 5-bromo-29-deoxyuridine (BrdU) (Sigma, UK) for 14 hours at 37uC in gelatinized Chamber slide (Nunc) After that, the cells were washed with PBS (Invitrogen) then fixed for 10 minutes in 4% paraformaldehyde. The fixed cells were incubated with 50 mM Glycine and then permibilized with 50 mM NaOH for 10 seconds, washed in PBS and blocked in 5% goat serum/ 0,1% NP-40/PBS for 30 minutes at room temperature. BrdU incorporation into DNA was detected with a monoclonal antibody anti BrdU G3–G4 (Sigma-Aldrich) diluted 1:500 at room temperature for 1 hour. Goat-anti mouse conjugated with FITC (Amersham) was used as secondary antiboby for immunofluores- cence detection. Nuclei were counterstaining with DAPI and visualized with a fluorescent microscope. Images were taken with a digital camera (Zeiss Axiocam 130061030 pixel) mounted on a Axioplan 2 microscope (Carl Zeiss Inc.) with 60x 1.40 objectives (Carl Zeiss Inc.) at room temperature using AxioVision software (Carl Zeiss Inc.). Cell Cycle Analysis Cellular DNA content was determined by staining cells with propidium iodide and analyzed them on a FACS instrument (Becton-Dickinson). ES cells were trypsinized, washed with PBS and resuspended in 500 ml of staining solution containing Na- citrate 0,1% (p/v), Nonidet P-40 0,1% (v/v), 10 mg/ml RNase and propidium iodide 50 mg/ml for 30 minutes. Cells were analyzed by FACS, and the proportion of cells in G0/G1, S, and G2/M phases was estimated by the Modfit cell-cycle analysis software. FACS Analysis Flow cytometry was performed according to Hao J et al. [35]. Briefly, EBs were dissociated into single cell suspensions after trypsinization (26106 cells). Following a wash with 10%FBS/ DMEM, cells were permeabilized with 0.05% saponin/PBS buffer for 20 minutes on ice. Cells were then stained with an antibody against sarcomeric myosin (MF-20 or cTnT antibodies, both from Hybridoma bank; dilution 1:100 in 10%FBS/DMEM) for 1 hour. PLoS ONE | www.plosone.org 10 February 2010 | Volume 5 | Issue 2 | e9029