title: Tetracycline-inducible gene expression in conditionally immortalized mouse podocytes.
abstract: BACKGROUND
  Conditionally immortalized podocytes are valuable research tools but are difficult to efficiently transfect and do not provide graded transgene expression.
  
  
  METHODS
  Conditionally immortalized mouse podocyte cell lines were established employing a tetracycline-inducible system. Glomerular cells, isolated from transgenic mice bear- ing two transgenes, NPHS2-reverse tetracycline-controlled transactivator, rtTA (A transgene) and H2-Kb-thermosensitive SV40 T, ts58A (I transgene), were cloned. One clone (AI podocytes) expressing WT1 and synaptopodin was transfected with pBI-EGFP (enhanced green fluorescent protein, G transgene) and separately with ptTS-Neo (transcriptional suppressor, T transgene) to produce stable transformants, AIG podocytes and AIT podocytes.
  
  
  RESULTS
  AIG podocytes expressed EGFP at 33 and 37 degrees C after doxycycline treatment, and retained podocin and rtTA mRNA expression and temperature-sensitive growth regulation. AIT podocytes, transiently transfected with luciferase-BI-EGFP (LG transgene), showed reduced background expression of EGFP and luciferase in the absence of doxycycline. In AITLG podocytes, generated by stable transfection of AIT podocytes with the LG transgene, luciferase expression was tightly regulated by doxycycline in a time- and concentration-dependent manner both at 33 and 37 degrees C, although background expression was not entirely eliminated. These podocytes retained temperature-sensitive growth regulation and expression of podocyte differentiation markers.
  
  
  CONCLUSIONS
  Mouse podocytes expressed tetracycline-induced transgenes efficiently while retaining differentiation markers.
authors.affiliation: Kidney Disease Section, Kidney Disease Branch, National Institute of Diabetes, Digestive and Kidney Diseases, Bethesda, MD 20892-1268, USA.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Kajiyama
authors.forename: Steve
authors.initials: S
authors.lastname: Titus
authors.forename: Christopher P
authors.initials: CP
authors.lastname: Austin
authors.forename: Kathleen
authors.initials: K
authors.lastname: Chiotos
authors.forename: Takayuki
authors.initials: T
authors.lastname: Matsumoto
authors.forename: Toru
authors.initials: T
authors.lastname: Sakairi
authors.forename: Jeffrey B
authors.initials: JB
authors.lastname: Kopp