Project description:TÜAB zebrafish were maintained at 28.5 °C in in 1x Danieau solution (58 mM NaCl, 0.7 mM KCl, 0.4 mM MgSO4, 0.6 mM Ca (NO3)2, 5 mM HEPES, pH 7.6, 0.0001 % Methylene blue). Morpholinos (Gene Tools, Philomath, OR) were designed against Danio rerio lin-9 homolog (Genebank accession NM_001044946). The morpholino (MO) sequences were MO-E1 5´-GTTAGTTTTATTACTCACTCTCGTC-3´ and 5-base mismatch morpholino MO-E1mis 5´-GTTACTTTTAATACTGACTGTCCTC-3´. 7 ng of morpholinos were injected into one cell-stage embryos. 20 embryos per condition (MO E1; MO E1mis) were pooled for RNA purification 24 h post fertilization. Using the two color Quick-Amp Labeling Kit (Agilent; 5190-0444) 100 ng of total RNA were used for cDNA synthesis, mRNA amplification and labeling according to manufacturers instructions. Transcriptional profiling was done on a zebrafish oligo array (Agilent; G2519F AMADID 019161) in a 4 x 44K slide format and analyzed as described before.<br>
Project description:Wild type (C57BL/6J) mice were divided in control (W), leptin-treated (E) and pair-fed (F). Obese (ob/ob) mice were divided in control (O), leptin-treated (L) and pair-fed (P). Control (W and O) and pair-fed animals (F and P) received vehicle (PBS), while E and L groups were intraperitoneally administrated with leptin for 28 days. Control (W and O) and leptin-treated (E and L) groups were provided with water and food ad libitum with, while daily food intake of pair fed (F and P) groups were matched to the amount eaten by the leptin-treated groups (E and L) the day before to discriminate the inhibitory effect of leptin on appetite.
Project description:The cell line "6780" derived from murine T-cell lymphoma was grown in RPMI medium 1640 with 10% FBS/1% penicillin/Streptomycin/1% l-glutamine/50 μM 2-mercaptoethanol. Vector constructs containing a dominant negative mutant of the human TGF-beta receptor type II (pMSCV-TGFBR-IRES-GFP) or an empty vector (pMSCV-IRES-GF) were kindly provided by Martin Eilers Group, IMT, Marburg, Germany. Cells were incubated with retroviruses containing supernatants for 12 h at 32°C in media containing 4 ?g/ml polybrene. Cells then were expanded at 37°C for an additional 48 h, and GFP-expressing cells were purified by flow cytometry on a FACS Vantage (Becton Dickinson).<br><br><br><br>All animal experiments were performed by following the guidelines from Administrative Panel on Laboratory Animal Care at Stanford University (protocol 8144). For transplantation experiment cells were washed once with PBS and 10x106 cells/site were injected subcutaneously (s.c.) into the flank of FVB/N syngeneic mice. Tumor growth was measured by using a caliper. To inactivate MYC expression and to assess for tumor regression, the drinking water was supplemented with 200 μg/ml doxycycline. Tumors were harvested untreated or after 1, 3 and 4 days upon doxycycline treatment, respectively. Total RNA was isolated from snap-frozen tumor tissue using the RNeasy Kit including DNase-I digest (Qiagen, Valencia, USA) following the manufacturer's protocol.
Project description:Seven temperature sensitive Saccharomyces cerevisiae BY4741 mutants (rsc3-1, abf1-101, reb1-212, rap1-1, mcm1, tbf1 and cep3-1) were grown at restrictive temperatures until a difference in OD600 was observed relative to a wild-type control. Nucleosomal DNA, whole genomic DNA and total RNA were isolated and hybridized onto yeast whole-genome tiling arrays.<br>
Project description:U2OS cells were stably transfected with an ecotropic receptor expression plasmid. These cells were infected with retroviruses expressing MIZ-1 or MYC and subsequently superinfected with retroviruses expressing p14ARF. Selection was carried out 48h following superinfection and selected cells were harvested within 1 - 2 passages. For each condition around 2.5x10<superscript6> cells were pooled.<br>Using the two color Quick-Amp labelling kit (Agilent, 5190-0444) 100ng of total RNA were used for cDNA synthesis, aRNA amplification and labelling according to manufacturer's instructions.<br>Transcriptional profiling was done on a whole human genome oligo microarray (Agilent, G4112F, 014850) in a 4x44k slide format.