Comment[ArrayExpressAccession] E-GEOD-58988 MAGE-TAB Version 1.1 Public Release Date 2014-12-23 Investigation Title Analyzing cold tolerance mechanism in transgenic zebrafish (Danio rerio) Comment[Submitted Name] Analyzing cold tolerance mechanism in transgenic zebrafish (Danio rerio) Experiment Description Low temperatures may cause severe growth inhibition and mortality in fish. In order to understand the mechanism of cold tolerance, a transgenic zebrafish Tg (smyd1:m3ck) model was established to study the effect of energy homeostasis during cold stress. The muscle-specific promoter Smyd1 was used to express the carp muscle form III of creatine kinase (M3-CK), which maintained enzymatic activity at a relatively low temperature, in zebrafish skeletal muscle. In situ hybridization showed that M3-CK was expressed strongly in the skeletal muscle. When exposed to 13M-BM-0C, Tg (smyd1:m3ck) fish maintained their swimming behavior, while the wild-type could not. Energy measurements showed that the concentration of ATP increased in Tg (smyd1:m3ck) versus wild-type fish at 28M-BM-0C. After 2 h at 13M-BM-0C, ATP concentrations were 2.16-fold higher in Tg (smyd1:m3ck) than in wild-type (P < 0.05). At 13M-BM-0C, the ATP concentration in Tg (smyd1:m3ck) fish and wild-type fish was 63.3% and 20.0%, respectively, of that in wild-type fish at 28M-BM-0C. Microarray analysis revealed differential expression of 1249 transcripts in Tg (smyd1:m3ck) versus wild-type fish under cold stress. Biological processes that were significantly overrepresented in this group included circadian rhythm, energy metabolism, lipid transport, and metabolism. These results are clues to understanding the mechanisms underlying temperature acclimation in fish. Gene expression in triplicate samples of m3ck-13M-BM-0C, m3ck-28M-BM-0C, wt-13M-BM-0C, and wt-28M-BM-0C was assessed. Twelve microarray experiments were performed, each with three fish. Term Source Name ArrayExpress EFO Term Source File http://www.ebi.ac.uk/arrayexpress/ http://www.ebi.ac.uk/efo/efo.owl Person Last Name Wang Wang Tan Jiao You Zhang Person First Name Qian Qian Xungang Shuang Feng Pei-Jun Person Email ameliaxing@163.com Person Affiliation Institute of Oceanology, Chinese Academy of Sciences Person Address Institute of Oceanology, Chinese Academy of Sciences, 7 Nanhai Road, Qingdao, China Person Roles submitter Protocol Name P-GSE58988-1 P-GSE58988-5 P-GSE58988-6 P-GSE58988-2 P-GSE58988-3 P-GSE58988-4 P-GSE58988-7 Protocol Description Data were extracted with Feature Extraction software 10.7 (Agilent technologies, Santa Clara, CA, USA). Raw data were normalized by Quantile algorithm, Gene Spring Software 11.0 (Agilent technologies, Santa Clara, CA, USA). ID_REF = VALUE = Normalized signal intensity Total RNA was amplified and labeled with Cy3-CTP by Low Input Quick Amp Labeling Kit, One-Color (Agilent technologies, Santa Clara, CA, USA), following the manufacturerM-bM-^@M-^Ys instructions. Labeled cRNA were purified by RNeasy mini kit (QIAGEN, GmBH, Germany). Each slide was hybridized with 1.65 M-5g Cy3-labeled cRNA using the Gene Expression Hybridization Kit (Agilent technologies Santa Clara, USA) in Hybridization Oven (Agilent technologies Santa Clara, USA). After 17 hours hybridization, slides were washed with Gene Expression Wash Buffer Kit (Agilent technologies Santa Clara, USA). The F2 generation was raised to the age of 3 months (2.17 M-1 0.02 cm, 0.42 M-1 0.01 g) before use in the cold acclamation experiment. 13M-0C was selected to represent cold stress. Cold stress was applied for 2 h according to previous study and our own preliminary experiments. Before cold treatment, the fish were transferred to tanks with small openings in the bottom and allowed to stand for one day. They were gently moved to the cold bath for the experiment. The groups (n = 30 per group) were designated m3ck-13M-0C, m3ck-28M-0C, wt-13M-0C, and wt-28M-0C. After temperature stress for 2 h, the fish were anesthetized with 100 mg/L MS222 and transferred immediately into liquid nitrogen. Wild-type and transgenic fish were reared at 28M-0C M-1 0.5M-0C and under a photoperiod regime of 14/10 h light/dark. Water quality was maintained by circulation with a filtration system. RNA samples were extracted from whole zebrafish with Trizol Reagent (Invitrogen, CA, USA). RNA quality as expressed by the RNA integrity number (RIN) was determined on an Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA) and then purified with an RNeasy mini kit (QIAGEN GmBH, Germany) with the RNase-Free DNase Set (QIAGEN). Slides were scanned using Agilent Microarray Scanner (Agilent technologies Santa C lara, USA) with default settings, Dye channel: Green, Scan resolution=5 M-NM-