Identification of Differentially Expressed Genes Between

J. Anim. Sci. & Technol. 51(6) 443~452, 2009 Identification of Differentially Expressed Genes Between Preadipocytes and Adipocytes Using Affymetrix Bovine Genome Array Seong Lan Yu*, Sang Mi Lee**, Man Jong Kang**, Hang Jin Jeong*, Byung Chan Sang*, Jin Tae Jeon*** and Jun Heon Lee* Division of Animal Science and Resources, College of Agriculture and Life Sciences, Chungnam National University*, Department of Animal Science, College of Agriculture and Life Science, Chonnam National University**, Division of Applied Life Science, Gyeongsang National University*** ABSTRACT Adipocytes are differentiated from preadipocytes and have large capacity for storing fats inside cells. In cattle, intramuscular fat (IMF) content is one of the major determinants for meat quality and also highly affects market prices, especially in Japan and Korea. In order to profiling differentially expressed genes between intramuscular fibroblast-like cells (preadipocytes) and their differentiated adipocytes, we have established intramuscular fibroblast-like cells from M. longissimus thoracis in Korean cattle (Hanwoo). The differentially expressed genes were selected by comparing these two types of cells ug thecommercially available 23kese two types of cells ug theco. The results indan ced that 206 arecomelements were differentially expressed. Of these, 67 and 94 ks wn genes were up and d wn regulaced, respectively, in adipocytes ug ng both 2-fold difference and Welch’s t-test as the cut-off points. The differentially expressed genes identified in this study can be used as good markers for improving meat quality traits with further verification of their biological functions, especially IMF contents in cattle. (Key words : Adipocytes, Bovine genome array, Differentially expressed genes, Intramuscular fat, Korean cattle) INTRODUCTION (C/EBP), peroxisome proliferator activated receptor-γ (PPARr) families, and adipocyte determination and differentiation Adipogenesis is the differentiation processes from the primitive dependent factor 1 (ADD1) (Rosen and Spiegelman, 2000; mesoderm stem cells to preadipocytes and ultimately become Rangwala and Lazar, 2000). Also, adipocyte differentiation was adipocytes. This process occurs at both prenatal and postnatal regulated by differentiation-inducing agents including insulin, states. The adipose tissues are very important because they can dexamethasone and methyl-isobutylxanthine (MIX; a phos- store fats as energy sources. Recently, researches on these cells phodiesterase inhibitor) (Girard et al., 1994; Wu et al., 1999; have been growing because the basic mechanisms of the adipose Reusch et al., 2000). tissue development can give the solutions for obesity, type II In case of cattle, many researches already have been carried out diabetes, hypertension and coronary heart diseases (Zalesin et al., for identifying underline mechanisms for intramuscular fat 2008). The basis of these researches was started by making deposition. The fat deposition inside muscles is called marbling immortal preadipocyte cell lines (Green and Kehinde, 1975). and this trait is one of the important factors for determining meat Later on, molecular regulation of adipocyte differentiation has quality in cattle (Wood et al., 1999). However, meat quality is been investigated using immortalized cell lines such as 3T3-L1, very complex trait because it includes all meat related factors Ob1771, and 3T3-F442A. The results were confirmed by in vivo containing tenderness, flavor, and juiciness. Aso et al. (1995) studies including knock out mice and/or transgenic mice (Rosen firstly established intramuscular preadipocyte (BIP) cell line in and Spiegelman, 2000). These results indicated that adipocyte cattle and they suggested that adipose tissue metabolism in differentiation was controlled by a number of adipogenic ruminants was different from that of non‐ruminants. In addition, transcription factors, including CCAAT-enhancer binding protein they found that extracellular matrix (ECM) of adipose tissue Corresponding author : Dr. J. H. Lee, Division of Animal Science & Resources, College of Agriculture and Life Sciences, Chungnam National University, Daejeon 305-764, Korea Tel: 82-42-821-5779, Fax: 82-42-825-9754, E-mail: [email protected] －443 － Yu et al. ; Differentially Expressed Genes in Adipocyte Differentiation during adipogenesis was newly organized and alternations of this was washed with 1 volume of 75% ethanol and briefly air‐dried. extracellular matrix were associated with ECM proteins (collagen, Total RNA was subjected to RNA clean up by RNeasyTM kit laminin and fibronectin) as well as caveolin-1 and cytoskeletal (QIAGEN, Germany). Total RNA integrity was monitored by proteins (actin and vimentin), which play important roles in denaturing 1% agarose gel electrophoresis. RNA concentrations intracellular accumulation of lipid droplets (Aso et al., 1995; and purities were measured by spectrophotometer (Amersham Nakajima et al., 1998; Inoue-Murayama et al., 2000; Nakajima et Pharmacia Biotech, UK). al., 2002; Tahara et al., 2004; Takenouchi et al., 2004). However, these results have limitations for only investigation of 3. GeneChip array analysis and data analysis extracellular matrix. Recently, Taniguchi et al. (2007) profiled differentially expressed genes during early adipogenic Microarray analyses were performed in two replicates differentiation of bovine perimuscular fat (PMF) preadipocytes. according to the Affymetrix GeneChip Expression Analysis In this study, we investigated differentially expressed genes Technical Manual (Affymetrix, USA). First strand cDNA was between intramuscular fibroblast‐like cells and their differentiated synthesized from reaction containing 10 μg total RNA, 50 μM adipocytes using 23k bovine genome array (Affymetrix, USA) in T7-Oligomer, 1× first-strand reaction buffer, 100 mM DTT, 500 order to identify important genes for adipogenesis and ultimately μM dNTP, and 200 U/μl Superscript II RTase (Gibco BRL, USA) give the basic information for identification of genes for the IMF at 42°C for 1 hour. To synthesis second strand of DNA, the first contents in cattle. strand DNA was added in the solution containing 5× second‐strand reaction buffer, 200 μM dNTP, 10 unit E. coli DNA MATERIALS AND METHODS ligase, 40 unit E. coli DNA polymerase I, and E. coli RNase H and reaction was carried out at 16°C for 2 hour. Double‐stranded 1. Preparation and Differentiation of intramuscular cDNA was purified using Sample Cleanup Module (Affymetrix, fibroblast-like cells USA) and labeled Biotin by in vitro transcription using Enzo Bioarray (Affymetrix, USA). The cDNA was hybridized with the The primary intramuscular fibroblast like cells were prepared solution containing 50 pM Oligo B2 (3 nM), 0.1 mg/ml herring and differentiated using the method described by Jeong et al. sperm DNA, 0.5 mg/ml acetylated BSA, 1× hybridization buffer, (2008). In order to confirm the cell differentiation, the cells were and 10% DMSO at 45°C for 16 hours. After hybridization, the stained with Oil Red O. The triglycerides (TG) in the arrays were washed in a GeneChip Fluidics station and stained differentiated cells were quantified using Triglyceride G Test Kit with a streptavidin-phycoerythrin complex. The intensities of chip (Wako, USA). All experiments were performed in triplicate. elements were determined with a GeneChip scanner. After scanning, data acquisition and image processing were carried out 2. Total RNA preparation with the GeneChip Operating Software (GCOS) and normalization of the data was done with MA‐plot. Finally, significant test was In order to isolate total RNA from intramuscular fibroblast‐like performed using Welch’s t‐test and Volcano Plot. In order to cells and their differentiated adipoctyes, 0.8 ml Tri Reagent identify differentially expressed genes in duplicate experiments, (Molecular Research Center, USA) was added to the cultured cells multiple test correction was also performed. The classification of in 60 mm dish and the cells were lysed by repetitive pipettings. gene ontology was performed using the Database for Annotation, The homogenates were stored at room temperature for 5 min for Visualization and Integrated Discovery (DAVID; http://david. the complete dissociation of nucleoprotein complexes. Then it was abcc.ncifcrf.gov/). vigorously mixed with 0.2 volume of chloroform and centrifuged at 12,000 rpm for 15 min at 4°C. The aqueous phase was 4. Quantitative real-time PCR transferred to a fresh tube and 0.5 volume of isopropanol was added. Then, the samples were left at room temperature for 10 Single‐stranded cDNA was synthesized from 1 μg total min, and centrifuged at 12,000 rpm for 8 min at 4°C. In order to RNA of intramuscular fibroblast-like cells and their obtain the RNA pellet, supernatant was removed and RNA pellet differentiated adipoctyes. Initially, RNA was denatured at 70ºC －444 － Yu et al. ; Differentially Expressed Genes in Adipocyte Differentiation for 10 min and reverse transcription was performed in 2. Identification of differentially expressed genes reactions containing 5 mM MgCl2, 10 mM Tris‐HCl (pH 9.0), ® 50 mM KCl, 0.1% Triton X‐100, 1 mM each dNTP, 0.5 unit The differentially expressed genes between intramuscular ® Recombinant RNasin Ribonuclease Inhibitor, 15 unit AMV fibroblast-like cells and their differentiated adipocytes were Reverse Transcriptase (Promega, USA) and 500 ng Oligo investigated using 23k Bovine Genome array (Affymetrix, USA). 15 (dT) primer at 42ºC for 1 hour. In order to identify differentially

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