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Isolation, culture and differentiation of duck (Anas platyrhynchos) preadipocytes.

ABSTRACT: In the present study, we isolated preadipocytes from the adipose tissue of Peking duck and subsequently cultured them in vitro. Cell counting kit-8 assay was employed to establish the growth curve of duck primary preadipocytes. Meanwhile, after the cells reaching full confluency, they were induced to differentiate into mature adipocytes by the addition of a cocktail containing dexamethasone, insulin, 3-isobutyl-1-methylxanthine, and oleic acid for 8 days. Successful differentiation was demonstrated by the development of lipid droplets and the expression of key marker genes including peroxisome proliferator-activated receptor-? (PPAR?), CCAAT/enhancer binding protein-? (CEBP/?) and adipocyte fatty acid-binding protein (FABP4). Our results showed that duck primary preadipocytes began to adhere 12 h after seeding as short spindle shapes or litter triangles, which grew quickly 3 days post attachment and maintained stable after day 7. After 8 days the preadipocytes were induced to differentiate into mature adipocytes, which were stained red by oil red O. Additionally, it showed that during preadipocyte differentiation PPAR? mRNA was highly expressed at day 3, while CEBP/? and FABP4 mRNA peaked at day 5 and 8, respectively. These results indicate that we have successfully isolated and cultured Peking duck preadipocytes and successfully induced them to differentiate into mature adipocytes. This work could lay a foundation for further research into waterfowl adipogenesis.

SUBMITTER: Ding F

PROVIDER: S-EPMC4545434 | biostudies-literature | 2015 Oct

REPOSITORIES: biostudies-literature

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Isolation, culture and differentiation of duck (Anas platyrhynchos) preadipocytes.

Ding Fang F Li Qing-Qing QQ Li Le L Gan Chao C Yuan Xin X Gou Hua H He Hua H Han Chun-Chun CC Wang Ji-Wen JW

Cytotechnology 20140403 5

In the present study, we isolated preadipocytes from the adipose tissue of Peking duck and subsequently cultured them in vitro. Cell counting kit-8 assay was employed to establish the growth curve of duck primary preadipocytes. Meanwhile, after the cells reaching full confluency, they were induced to differentiate into mature adipocytes by the addition of a cocktail containing dexamethasone, insulin, 3-isobutyl-1-methylxanthine, and oleic acid for 8 days. Successful differentiation was demonstra ...[more]

PMID: 24696190

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Project description:Background: Skeletal muscle growth and development is highly orchestrated in gene expressions. Understanding the location-specific and breed-specific genes and pathways are agriculturally important on meat products and meat quality. In the present study, RNA-Seq was performed to elucidate the difference of muscle deposition between locations and breeds for functional genomics studies. To achieve those goals, skeletal muscle samples were collected for pectoral muscle (PM) and leg muscle (LM) of two genetically different duck breeds, Heiwu duck (H) and Peking duck (P), at embryonic 15 days. Functional genomics studies were performed in two experiments: Experiment 1 directly compared location-specific genes between PM and LM, while Experiment 2 compared the two breeds (H and P) at the same development stage (embryonic 15 days). Results: Almost 13 million clean reads was generated using Illumina technology on each library, and more than 70% reads mapped to Anas platyrhynchos breed Peking duck genome. We identified 246, 258, 20 and 54 differentially expressed genes (DEGs) (log2.fold-change >0.5, and P-value <0.05) for H-PM vs H-LM, P-PM vs P-LM, H-LM vs P-LM and H-PM vs P-PM library, separately. A total of 168 genes were differentially expressed by over locations in experiment 1, while only 8 genes were differentially expressed by over breeds in experiment 2. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes pathways (KEGG) were used to functionally annotate DGEs. In experiment 1, DEGs mainly involved in Focal adhesion, PI3K-Akt signaling pathway and ECM-receptor interaction pathways (corrected P-value<0.05). In experiment 2, DEGs only associated with ribosome signal pathway (corrected P-value<0.05). In addition, quantitative real-time PCR was employed to confirm 15 differentially expressed genes detected by RNA-Seq. Conclusions: Comparative transcript analysis of leg muscle and pectoral muscle of two duck breeds not only improves our understanding of those location-specific and breed-specific genes and pathways, but also provides some candidate molecular targets for increase muscle products and meat quality by genetic control. Conclusions: Our study represents the first detailed analysis of duck breed muscle transcriptomes generated by RNA-seq technology. The optimized data analysis workflows reported here should provide a framework for comparative investigations of expression profiles. Our results show that NGS offers a comprehensive and more accurate quantitative and qualitative evaluation of mRNA content within a cell or tissue. We conclude that RNA-seq based transcriptome characterization would expedite genetic network analyses and permit the dissection of complex biologic functions.

Dataset Information

Isolation, culture and differentiation of duck (Anas platyrhynchos) preadipocytes.

Publications

Isolation, culture and differentiation of duck (Anas platyrhynchos) preadipocytes.

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