title: [Establishment of an albumin and cytokeratin 19 genetically-modified embryonic stem cell line and evaluation of its hepatoblast differentiation capacities].
abstract: OBJECTIVE
  To establish a gene-modified embryonic stem (ES; E14.1-2) cell line with hepatoblast differentiation reporter genes, albumin (ALB) and cytokeratin 19 (CK19), labeled to facilitate study of their potential applicability as differentiated hepatoblasts.
  
  
  METHODS
  Two expression vectors were constructed, one with the ALB promotor driving the enhanced green fluorescent protein (EGFP) and anti-neomycin genes (pAlb-EGFP), and the other with the CK19 promotor driving the red fluorescence protein and anti-hygromycin genes (pCK19-hCD25-IRES-tdTOMATO). The linearized vectors were electroporated into the E14.1 line, and double reporter genes-modified ES cells (E14.1-2) were selected by neomycin and hygromycin. E14.1-2 hepatoblast differentiation was induced by exposure to growth factors (BMP4 and bFGF) and evidenced by embryoid body formation. Fluorescence-activated cell sorting (FACS) and reverse transcription-polymerase chain reaction (RT-PCR) were used to confirm whether differentiated cells were hepatoblast-like and to quantify the differentiation efficiency.
  
  
  RESULTS
  The pAlb-EGFP and pCK19-hCD25-IRES-tdTOMATO vectors were shown to specifically activate ALB and CK19 expression. The E14.1-2 cell line with labeled ALB and CK19 was established, and shown to have pluripotency by RT-PCR detection of pluripotent markers' expression, namely Oct4 and SSEA-1. After 22 days of induction, 21.27% of the differentiated hepatoblasts were detected by FACS as positive for ALB and CK19 expression.
  
  
  CONCLUSIONS
  A gene-modified ES cell line was generated with hepatocyte differentiation reporter genes ALB and CK19 labeled. The differentiation of the resultant E14.1-2 line was technically simple to qualify and quantify, and will likely aid future studies of hepatoblast characteristics.
authors.affiliation: Department of General Surgery, Beijing Hospital, Beijing 100730, China.
authors.forename: Yong
authors.initials: Y
authors.lastname: Lan
authors.forename: Yang-fang
authors.initials: YF
authors.lastname: Li
authors.forename: Da-jun
authors.initials: DJ
authors.lastname: Li
authors.forename: Jun-min
authors.initials: JM
authors.lastname: Wei
authors.forename: Xin
authors.initials: X
authors.lastname: Wang
authors.forename: He-ping
authors.initials: HP
authors.lastname: Hu
mesh_terms: D000418:Albumins / Q000235:genetics; D000818:Animals; D015415:Biomarkers; D002454:Cell Differentiation; D002460:Cell Line; D053595:Embryonic Stem Cells / Q000166:cytology*; D017930:Genes, Reporter; D022781:Hepatocytes / Q000166:cytology*; D053539:Keratin-19 / Q000235:genetics; D051379:Mice; D014162:Transfection
chemical_list: D000418:Albumins; D015415:Biomarkers; D053539:Keratin-19