Tetracycline-inducible gene expression in conditionally immortalized mouse podocytes.
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.
Kajiyama
Hiroshi
H
Kidney Disease Section, Kidney Disease Branch, National Institute of Diabetes, Digestive and Kidney Diseases, Bethesda, MD 20892-1268, USA.
Titus
Steve
S
Austin
Christopher P
CP
Chiotos
Kathleen
K
Matsumoto
Takayuki
T
Sakairi
Toru
T
Kopp
Jeffrey B
JB