Immortalization of chicken preadipocytes by retroviral transduction of chicken TERT and TR

Abstract

The chicken is an important agricultural animal and model for developmental biology, immunology and virology. Excess fat accumulation continues to be a serious problem for the chicken industry. However, chicken adipogenesis and obesity have not been well investigated, because no chicken preadipocyte cell lines have been generated thus far. Here, we successfully generated two immortalized chicken preadipocyte cell lines through transduction of either chicken telomerase reverse transcriptase (chTERT) alone or in combination with chicken telomerase RNA (chTR). Both of these cell lines have survived >100 population doublings in vitro, display high telomerase activity and have no sign of replicative senescence. Similar to primary chicken preadipocytes, these two cell lines display a fibroblast-like morphology, retain the capacity to differentiate into adipocytes, and do not display any signs of malignant transformation. Isoenzyme analysis and PCR-based analysis confirmed that these two cell lines are of chicken origin and are free from inter-species contamination. To our knowledge, this is the first report demonstrating the generation of immortal chicken cells by introduction of chTERT and chTR. Our established chicken preadipocyte cell lines show great promise as an in vitro model for the investigation of chicken adipogenesis, lipid metabolism, and obesity and its related diseases, and our results also provide clues for immortalizing other avian cell types.

MiR-17-5p and miR-20a promote chicken cell proliferation at least in part by upregulation of c-Myc via MAP3K2 targeting

“…DF1 cells were purchased from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. Immortalized chicken preadipocytes (ICPA-1) were generated in the lab 59 .…”

An Upstream Open Reading Frame Represses Translation of Chicken PPARγ Transcript Variant 1

“…Accumulating evidence shows that adipogenesis and lipid metabolism are different between mammals and chickens (Prigge and Grande, 1971;Ji et al, 2012). For example, unlike that in mammals, chicken adipocyte lipolysis is almost exclusively regulated by glucagon, and chicken adipose tissue is not sensitive to insulin (Dupont et al, 2012;Ji et al, 2012;Wang et al, 2017). In the present study, we demonstrated that the uORF represses chicken PPARγ1 translation, and bioinformatics analysis showed that the 5 UTR sequences of PPARγ transcripts had low sequence similarity among humans, mice, and chickens, and no uORF element was identified in the 5 UTRs of human and mouse PPARγ transcripts.…”

“…Chicken embryo fibroblast (DF1) cell line was purchased from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, and the immortalized chicken preadipocyte cell line 1 (ICP1) was generated in our laboratory (Wang et al, 2017). All cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin, at 37 • C and 5% CO 2 .…”

An upstream open reading frame represses translation of chicken PPARγ transcript variant 1

“…Accumulating evidence shows that adipogenesis and lipid metabolism are different between mammals and chickens (Prigge and Grande 1971;Ji et al 2012). For example, unlike that in mammals, chicken adipocyte lipolysis is almost exclusively regulated by glucagon, and chicken adipose tissue is not sensitive to insulin (Dupont et al 2012;Ji et al 2012;Wang et al 2017 Compared with the wild-type PPARγ1 5′ UTR reporter, the luciferase activity of Translational inhibition of PPARγ1 may be due to uORF-mediated ribosome stalling (A) or inefficient reinitiation at the authentic start codon of PPARγ1 (B).…”

“…Cell culture DF1 cells were purchased from the Institute of Biochemistry and Cell Biology, 5 Chinese Academy of Sciences, and the immortalized chicken preadipocyte cell line 1 (ICP1) was generated in our laboratory (Wang et al 2017). All cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin, at 37 °C and 5% CO 2 .…”