Scientia Agricultura Sinica ›› 2015, Vol. 48 ›› Issue (8): 1624-1631.doi: 10.3864/j.issn.0578-1752.2015.08.17

• ANIMAL SCIENCE·VETERINARY SCIENCERE • Previous Articles     Next Articles

Effect of HOPX Gene Overexpression on Chicken Preadipocyte Proliferation

SHI Hong-yan, HE Qi, CHENG Min, SUN Ying-ning, LI Hui, WANG Ning   

  1. College of Animal Science and Technology, Northeast Agricultural University/Key Laboratory of Chicken Genetics and Breeding, Ministry of Agriculture/Key Laboratory of Animal Genetics, Breeding and Reproduction, Education Department of Heilongjiang Province, Harbin 150030
  • Received:2014-03-13 Online:2015-04-16 Published:2015-04-16

Abstract: 【Objective】The objective of this study was to construct the eukaryotic expression vector of chicken full-length HOPX gene and investigate the effect of HOPX gene overexpression on chicken preadipocytes proliferation.【Method】Using Primer Premier 5.0 software, a pair of primers was designed to amplify the full-length coding sequence (CDS) of chicken HOPX. The full-length coding sequence (CDS) of chicken HOPX gene was PCR amplified from the cDNA from the abdominal fat tissues of AA broiler chickens and cloned into pCMV-HA vector. Chicken preadipocytes were isolated from the abdominal fat tissues of 12-day-old AA broiler chicken by collagenase digestion, cultured and transfected with pCMV-HA-HOPX and pCMV-HA empty vector, respectively. Cell proliferation was assayed by microscopic examination and Cell Counting Kit-8 (CCK-8). Gene expression was measured by Western blotting and quantitative Real-time RT-PCR. 【Result】 The sequencing results showed that the full-length coding sequence of chicken HOPX gene is222bp and identical with NCBI reference sequence (NM_204556). Western blotting analysis showed that pCMV-HA-HOPX could correctly express the HA-tagged HOPX. The microscopic examination showed that the numbers of the preadipocytes transfected with pCMV-HA-HOPX were less than those of the preadipocytes transfected with empty pCMV-HA vector at 24 h and 48 h after of cell adhesion. CCK-8 analysis showed that the OD values of the preadipocytes transfected with pCMV-HA-HOPX were significantly lower than those of the preadipocytes transfected with empty pCMV-HA vector at 24 h, 48 h and 72 h after of cell adhesion (P0.01). Consistently, at 24 h after of cell adhesion, the mRNA expression of CyclinD1 was significantly lower in HOPX-overexpressing preadipocytes than in control preadipocytes (P<0.05); at 48 h after of cell adhesion, the mRNA expression of PCNA was significantly lower in HOPX-overexpressing preadipocytes than in control preadipocytes (P<0.05); at 72 h after of cell adhension, the mRNA expression of CyclinD1 and PCNA were extremely significantly lower in HOPX-overexpressing preadipocytes than in control preadipocytes (P<0.01) 【Conclusion】 HOPX gene overexpression in vitro inhibits chicken preadipocyte proliferation.

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