title: Recombinant adenovirus-mediated expression of GHS-R1a in HEK 293 cells.
abstract: OBJECTIVE
  To construct the recombinant adenovirus vector carrying human growth hormone secretagogue receptor type 1a (GHS-R1a), for genetic transfection.
  
  
  METHODS
  The full-length human GHS-R1a gene was obtained by PCR amplification and then cloned into the shuttle plasmid pAdTrack-CMV. The linearized plasmid pAdTrack-CMV-GHS-R1a was co-transformed into Escherichia coli (E. coli) BJ5183 cells along with an adenoviral backbone plasmid pAdEasy1. The HEK293 cells were then infected with adenoviruses. The expression of GHS-R1a was indicated by green fluorescent protein (GFP), and confirmed by Reverse Transcription Polymerase Chain Reaction (RT-PCR) and Western blot.
  
  
  RESULTS
  Enzymatic digestion of pAdGHS-R1a yielded a large fragment (approximately 30 kb) and a small fragment (4.5 kb), indicating the successful construction of recombinant adenovirus expression vector. Expression of GFP was observed by confocal laser scanning microscopy at 24 h after infection. RT-PCR and Western blot further confirmed that GHS-R1a was efficiently expressed in 293 cells.
  
  
  CONCLUSIONS
  Recombinant adenovirus (AdGHS-R1a) is successfully constructed, and the target gene can be expressed efficiently in 293 cells, which provide a valuable tool for further studying the function of GHS-R1a.
authors.affiliation: Department of Physiology, Shandong Provincial Key Laboratory of Pathogenesis and Prevention of Neurological Disorders and State Key Disciplines: Physiology, Medical College of Qingdao University, Qingdao 266071, China.
authors.forename: Li
authors.initials: L
authors.lastname: Liu
authors.forename: Hua-Min
authors.initials: HM
authors.lastname: Xu
authors.forename: Hong
authors.initials: H
authors.lastname: Jiang
authors.forename: Jun
authors.initials: J
authors.lastname: Wang
authors.forename: Ning
authors.initials: N
authors.lastname: Song
authors.forename: Jun-Xia
authors.initials: JX
authors.lastname: Xie
mesh_terms: D000256:Adenoviridae / Q000235:genetics*; D002460:Cell Line; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D054440:Receptors, Ghrelin / Q000235:genetics* / Q000378:metabolism*; D014162:Transfection
chemical_list: D054440:Receptors, Ghrelin; D049452:Green Fluorescent Proteins