Promoter structures and differential responses to viral and non-viral inducers of chicken melanoma differentiation-associated gene 5.
Melanoma differentiation-associated gene 5 (MDA5) is a member of the retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) family and plays a pivotal role in the anti-viral innate immune response. As RIG-I is absent in chickens, MDA5 is hypothesized to be important in detecting viral nucleic acids in the cytoplasm. However, the molecular mechanism of the regulation of chicken MDA5 (chMDA5) expression has yet to be fully elucidated. With this in mind, a ∼2.5kb chMDA5 gene promoter region was examined and PCR amplified to assess its role in immune response. A chMDA5 promoter reporter plasmid (piggybac-MDA5-DsRed) was constructed and transfected into DF-1 cells to establish a Piggybac-MDA5-DsRed cell line. The MDA5 promoter activity was extremely low under basal condition, but was dramatically increased when cells were stimulated with polyinosinic: polycytidylic acid (poly I:C), interferon beta (IFN-β) or Infectious Bursal Disease Virus (IBDV). The DsRed mRNA level represented the promoter activity and was remarkably increased, which matched the expression of endogenous MDA5. However, Infectious Bronchitis Virus (IBV) and Newcastle disease virus (NDV) failed to increase the MDA5 promoter activity and the expression of endogenous MDA5. The results indicated that the promoter and the Piggybac-MDA5-DsRed cell line could be utilized to determine whether a ligand regulates MDA5 expression. For the first time, this study provides a tool for testing chMDA5 expression and regulation.
State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, Guangxi High Education Laboratory for Animal Reproduction and Biotechnology, Guangxi University, Nanning, Guangxi, China.
Wen-Xin
WX
Zhang
State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, Guangxi High Education Laboratory for Animal Reproduction and Biotechnology, Guangxi University, Nanning, Guangxi, China.
Er-Wei
EW
Zuo
Guangxi Key Laboratory of Animal Vaccines and New Technology, Guangxi Veterinary Research Institute, Nanning, Guangxi, China; College of Animal Science and Technology, Guangxi University, Nanning, Guangxi, China.
Ying
Y
He
State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, Guangxi High Education Laboratory for Animal Reproduction and Biotechnology, Guangxi University, Nanning, Guangxi, China.
Dong-Yang
DY
Chen
State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, Guangxi High Education Laboratory for Animal Reproduction and Biotechnology, Guangxi University, Nanning, Guangxi, China.
Xie
X
Long
State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, Guangxi High Education Laboratory for Animal Reproduction and Biotechnology, Guangxi University, Nanning, Guangxi, China.
Mei-Juan
MJ
Chen
State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, Guangxi High Education Laboratory for Animal Reproduction and Biotechnology, Guangxi University, Nanning, Guangxi, China.
Ting-Ting
TT
Li
State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, Guangxi High Education Laboratory for Animal Reproduction and Biotechnology, Guangxi University, Nanning, Guangxi, China.
Xiao-Gan
XG
Yang
State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, Guangxi High Education Laboratory for Animal Reproduction and Biotechnology, Guangxi University, Nanning, Guangxi, China.
Hui-Yan
HY
Xu
State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, Guangxi High Education Laboratory for Animal Reproduction and Biotechnology, Guangxi University, Nanning, Guangxi, China.
Sheng-Sheng
SS
Lu
State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, Guangxi High Education Laboratory for Animal Reproduction and Biotechnology, Guangxi University, Nanning, Guangxi, China.
Ming
M
Zhang
State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, Guangxi High Education Laboratory for Animal Reproduction and Biotechnology, Guangxi University, Nanning, Guangxi, China.
Ke-Huan
KH
Lu
State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, Guangxi High Education Laboratory for Animal Reproduction and Biotechnology, Guangxi University, Nanning, Guangxi, China. Electronic address: luy800@gmail.com.
Yang-Qing
YQ
Lu
D000818:Animals; D002645:Chickens / Q000235:genetics / Q000276:immunology*; D005434:Flow Cytometry; D005786:Gene Expression Regulation / Q000276:immunology*; D007113:Immunity, Innate / Q000276:immunology*; D007243:Infectious bursal disease virus / Q000276:immunology; D000072640:Interferon-Induced Helicase, IFIH1 / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology; D011070:Poly I-C / Q000276:immunology; D011401:Promoter Regions, Genetic / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction
D000072640:Interferon-Induced Helicase, IFIH1; D011070:Poly I-C
Chicken; Innate immunology; MDA5; Promoter; RIG-I-like receptor (RLR); Virus