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Publication
Genome Engineering Renal Epithelial Cells for Enhanced Volume Transport
Function.
Authors Wilson MH, Veach RA, Luo W, Welch RC, Roy S, Fissell WH
Submitted By Submitted Externally on 2/12/2020
Status Published
Journal Cellular and molecular bioengineering
Year 2020
Date Published 2/1/2020
Volume : Pages 13 : 17 - 26
PubMed Reference 32030105
Abstract Bioengineering an implantable artificial kidney (IAK) will require renal
epithelial cells capable of reabsorption of salt and water. We used genome
engineering to modify cells for improved Na+/H+ exchange and H2O reabsorption.
The non-viral piggyBac transposon system enables genome engineering cells to
stably overexpress one or more transgenes simultaneously., We generated
epitope-tagged human sodium hydrogen exchanger 3 (NHE3) and aquaporin-1 (AQP1)
cDNA expressing piggyBac transposon vectors. Transgene expression was evaluated
via western blot and immunofluorescence. Flow cytometry analysis was used to
quantitate transporter expression in a library of genome engineered clones. Cell
surface biotinylation was used evaluate surface protein localization. Blister
formation assays were used to monitor cellular volumetric transport., piggyBac
enabled stable transposon integration and overexpression of cumate-inducible
NHE3 and/or constitutively expressing AQP1 in cultured renal (MDCK) epithelial
cells. Cell surface delivery of NHE3 and AQP1 was confirmed using cell surface
biotinylation assays. Flow cytometry of a library of MDCK clones revealed
varying expression of AQP1 and NHE3. MDCK cells expressing AQP1 and
cumate-inducible NHE3 demonstrated increased volumetric transport., Our results
demonstrate that renal epithelial cells an be genome engineered for enhanced
volumetric transport that will be needed for an IAK device. Our results lay the
foundation for future studies of genome engineering human kidney cells for renal
tubule cell therapy.





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