title: [Establishment of the cell line that human lung adenocarcinoma can stably express luciferase which is absent of nm23-H1 expression and detecting its luminescence in vitro and in vivo].
abstract: OBJECTIVE
  On the condition that laboratory animals survive, we can detect the distribution of tumor cells by in vivo imaging that were labeled with firefly- luciferase (luc) gene. The purpose of this study is to establish a light-emitting cell line A549/nm23-H1-shRNA-luc which could express nm23-H1 shRNA and firefly-luciferase stably, and detect its bioluminescence in vitro and in vivo. It will provide the preparation for the next related experimental research in vivo.
  
  
  METHODS
  The optimal concentration of hygromycin B for screening A549/nm23-H1-shRNA cells was determined by concentration gradient method. We firstly transfected the plasmid (PGL4.50) with luc gene into A549/nm23-H1-shRNA cells and then screened the monoclonal cell line A549/nm23-H1-shRNA-luc with hyhromycin B. The positive monoclonal cell line was identified with an in vivo imaging system, thereafter the expression stability of luciferase was analyzed in the strongest light-emitting positive monoclonal cell line. The A549/nm23-H1-shRNA-luc cells were inoculated subcutaneously into right-hind groin of nude mice and then observed by the in vivo imaging system.
  
  
  RESULTS
  The optimal concentration of hygromycin B used in screening A549/nm23-H1-shRNA cells was 300 μg/mL. After screening, the A549/nm23-H1-shRNA-luc cells established can express luciferase stably in vitro, a great linear correlation existed between the amount of cells (x) and bioluminescence values (y), with an equation of y=3,699.9x+992,237, and the square of the correlation coefficient (R2) was 0.975,1. To evaluate the stability of bioluminescence in vivo, 10 nude mice were randomly divided into two groups that the same number of cells were implanted into. The variation of bioluminescence values detected in vivo between the two groups of the same cells was not statistically significant (P>0.05).
  
  
  CONCLUSIONS
  We have successfully established the cell line A549/nm23-H1-shRNA-luc which can express luciferase persistently and stably.
authors.affiliation: Tianjin Key Laboratory of Lung Cancer Metastasis and Tumor Microenvironment, Tianjin Lung Cancer Institute, Tianjin Medical University General Hospital, Tianjin 300052, China.
authors.forename: Hongming
authors.initials: H
authors.lastname: Wang
authors.forename: Daxing
authors.initials: D
authors.lastname: Zhu
authors.forename: Zhihao
authors.initials: Z
authors.lastname: Wu
authors.forename: Qinghua
authors.initials: Q
authors.lastname: Zhou
mesh_terms: D000230:Adenocarcinoma / Q000737:chemistry / Q000201:enzymology / Q000235:genetics*; D000077192:Adenocarcinoma of Lung; D000818:Animals; D045744:Cell Line, Tumor / Q000737:chemistry* / Q000201:enzymology; D058948:Cell Tracking / Q000295:instrumentation / Q000379:methods*; D005260:Female; D015870:Gene Expression; D020868:Gene Silencing; D006801:Humans; D008156:Luciferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D049449:Luminescence; D008175:Lung Neoplasms / Q000737:chemistry / Q000201:enzymology / Q000235:genetics*; D051379:Mice; D008819:Mice, Nude; D054778:NM23 Nucleoside Diphosphate Kinases / Q000235:genetics* / Q000378:metabolism; D014162:Transfection
chemical_list: D054778:NM23 Nucleoside Diphosphate Kinases; D008156:Luciferases; C517938:NME1 protein, human