Establishment And Analysis Of Immortalized Chicken Preadipocyte Cell Lines

Chicken is an important non-mammalian model animal for developmental,virology and immunology studies.Because chicken is naturally hyperglycemia and insulin resistant,making it a potential model for studies on human obesity,insulin resistance and type 2 diabetes.Excessive fat accumulation(obesity)is an economic and environmental concern for the broiler industry,and urgently needs to be addressed.To solve this problem,it is necessary to gain insight into the molecular mechanisms of chicken adipocyte differentiation or adipogenesis.Unfortunately,so far no chicken preadipocyte cell lines have been generated,resulting in poor understanding of the molecular mechanisms underlying chicken adipogenesis and obesity.In this respect,the aim of this study was to immortalize chicken preadipocyte cells and reveal the gene expression profiling during chicken adipocyte differentiation.Here,for the first time,the chicken telomerase activity was restored by chicken telomerase reverse transcriptase(chTERT)and chicken telomerase RNA(chTR)and two immortalized chicken preadipocyte cell lines were established.Firstly,the chTERT and chTR were cloned,and their recombinant retrovirus vectors(pLXRN-chTERT and pLPCX-chTR)were constructed and cotransfected with pVSV-G into GP2-293 cells to prepare their respective retroviruses.Chicken preadipocytes were isolated and infected with either pLXRN-chTERT retrovirus alone or a combination of pLXRN-chTERT and pLPCX-chTR viruses,and selected for immortalized cells with antibiotics.Finally,two immortalized chicken preadipocyte cell lines were established through transduction of either chTERT alone,or in combination with chTR.The results demonstrated that chTERT,but not chTR,was the rate-limiting factor for chicken telomerase activity.The chTERT retrovirus-infected preadipocytes was designated as the immortalized chicken preadipocyte 1(ICP1),and the chTERT and chTR retrovirus-coinfected preadipocytes was designated as the immortalized chicken preadipocyte 2(ICP2).Both ICP1 and ICP2 had survived >100 population doublings in vitro,fulfilling the criteria defining immortalized cells.Cellular morphological examination showed that ICP1 and ICP2 displayed a fibroblast-like shape,and ?-gal staining showed that they had no signs of replicative senescence.Semi-quantitative RT-PCR and TRAP assay showed that these two cell lines expressed high levels of telomerase genes and gained high telomerase activity.DNA ploidy analysis indicated that these two cell lines were aneuploid,but the anchorage-independent growth assay showed that these two cell lines were not malignant transformed.Isoenzyme analysis and the PCR based analysis confirmed that ICP1 and ICP2 were authentic chicken cell lines and free from inter-species contamination.Similar to primary chicken preadipocytes,these two cell lines retained the capacity to differentiate to adipocytes,as demonstrated by Oil Red O staining and quantitative real-time RT-PCR of the adipocyte differentiation marker genes.Finally,the gene expression profiling during the ICP2 differentiation were analyzed by agilent chicken gene expression microarray(4×44K),and the differential expression genes(DEGs)between the two cell lines and the DEGs during chicken preadipocyte differentiation were identified and analyzed by GO and KEGG.To our knowledge,this is the first report demonstrating the generation of immortal chicken cells by introduction of chTERT alone or a combination of chTERT and chTR.Our established chicken preadipocyte cell lines show great promise as an in vitro model for investigation of chicken adipogenesis,lipid metabolism,obesity and its related diseases,and our results also provide clues for immortalizing other avian cell types.Our results of the gene expression profiling of chicken preadipocyte differentiation lay a solid foundation for unraveling the molecular mechanism underlying chicken adipocyte differentiation.