title: Construction of Gpm6a/ReelinGFPCreERT2by BAC recombination using a specific gene in hepatic mesothelial or stellate cells.
abstract: OBJECTIVE
  To prepare a Gpm6a/ReelinGFPCreERT2construct with a rapid and reliable strategy using a bacterial artificial chromosome (BAC).
  
  
  METHODS
  Gpm6a and Reelin BACs were purified and transformed into SW102E. coliby electroporation. The GFPCreERT2 fragment was prepared from a shuttle vector and transformed into SW102E. colicarrying a BAC. Homologous recombination was induced in SW102E. coli. Recombinant clones were screened and confirmed by PCR and restriction enzyme digestion. Recombinant clones were transformed into SW102E. colito remove the kanamycin unit.
  
  
  RESULTS
  A complete BAC was successfully transformed into SW102E. coliby electroporation because BAC purified from SW102E. colishowed the same pattern as the original BAC withBamHI digestion. The GFPCreERT2 fragment was deemed to have been prepared successfully because we obtained the same size fragment as expected. Homologous recombination was induced, and GFPCreERT2 was deemed to have been inserted into the correct site of the BAC because we found the band change was the same as the expected pattern after restriction enzyme digestion. The kanamycin unit was deemed to have been removed successfully because we obtained different sizes of bands that were consistent with the results expected by PCR with different primers.
  
  
  CONCLUSIONS
  The construct of Gpm6aGFPCreERT2or ReelinGFPCreERT2was prepared successfully, which will establish a foundation for tracing the hepatic stellate cell lineage and studying its function.
authors.affiliation: Hong-Bo Shi, Hong-Lin Shi, Feng Ren, Zhong-Ping Duan, Beijing Institute of Hepatology, Beijing Youan Hospital, Capital Medical University, Beijing 100069, China.
authors.forename: Hong-Bo
authors.initials: HB
authors.lastname: Shi
authors.affiliation: Hong-Bo Shi, Hong-Lin Shi, Feng Ren, Zhong-Ping Duan, Beijing Institute of Hepatology, Beijing Youan Hospital, Capital Medical University, Beijing 100069, China.
authors.forename: Jin-Li
authors.initials: JL
authors.lastname: Lou
authors.affiliation: Hong-Bo Shi, Hong-Lin Shi, Feng Ren, Zhong-Ping Duan, Beijing Institute of Hepatology, Beijing Youan Hospital, Capital Medical University, Beijing 100069, China.
authors.forename: Hong-Lin
authors.initials: HL
authors.lastname: Shi
authors.affiliation: Hong-Bo Shi, Hong-Lin Shi, Feng Ren, Zhong-Ping Duan, Beijing Institute of Hepatology, Beijing Youan Hospital, Capital Medical University, Beijing 100069, China.
authors.forename: Feng
authors.initials: F
authors.lastname: Ren
authors.affiliation: Hong-Bo Shi, Hong-Lin Shi, Feng Ren, Zhong-Ping Duan, Beijing Institute of Hepatology, Beijing Youan Hospital, Capital Medical University, Beijing 100069, China.
authors.forename: Yu
authors.initials: Y
authors.lastname: Chen
authors.affiliation: Hong-Bo Shi, Hong-Lin Shi, Feng Ren, Zhong-Ping Duan, Beijing Institute of Hepatology, Beijing Youan Hospital, Capital Medical University, Beijing 100069, China.
authors.forename: Zhong-Ping
authors.initials: ZP
authors.lastname: Duan
mesh_terms: D000818:Animals; D015816:Cell Adhesion Molecules, Neuronal / Q000235:genetics*; D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics*; D003001:Cloning, Molecular / Q000379:methods*; D018274:Electroporation; D004926:Escherichia coli / Q000235:genetics; D016326:Extracellular Matrix Proteins / Q000235:genetics*; D005260:Female; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000235:genetics; D055166:Hepatic Stellate Cells; D059765:Homologous Recombination; D019426:Integrases / Q000378:metabolism; D008099:Liver / Q000166:cytology; D008562:Membrane Glycoproteins / Q000235:genetics*; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D009419:Nerve Tissue Proteins / Q000235:genetics*; D016133:Polymerase Chain Reaction; D000091042:Reelin Protein; D012697:Serine Endopeptidases / Q000235:genetics*
chemical_list: D015816:Cell Adhesion Molecules, Neuronal; D016326:Extracellular Matrix Proteins; C484158:GPM6A protein, human; D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; D000091042:Reelin Protein; C045073:Cre recombinase; D019426:Integrases; C000717127:RELN protein, human; C000717128:Reln protein, mouse; D012697:Serine Endopeptidases
keywords: Bacterial artificial chromosome; Glycoprotein M6a; Homologous recombination; Reelin