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J. Contreras, K. Wu, A. Hseuh, S. Hamm-Alvarez; Efficient Transfection of Primary Rabbit Lacrimal Gland Acinar Cells by Nucleofection. Invest. Ophthalmol. Vis. Sci. 2009;50(13):3640.
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Rabbit lacrimal gland acinar cells (LGACs) are primary epithelial secretory cells that have previously shown very low transfection efficiency using non-viral, lipid-based methods. Here we investigated and optimized the efficiency of transfection using nucleofection for plasmid delivery using genes encoding cytosolic and membrane-encapsulated proteins, GFP and cathepsin-GFP, respectively.
Rabbit LGACs were isolated according to established protocols and cultured for 1-2 days prior to electroporation using the Amaxa (Lonza Group) nucleofector device and plasmid DNA (3 µg). The first plasmid used was Amaxa’s pmaxGFP control plasmid (3.4Kb). The second was a cathepsin-GFP fusion construct made by using the pAcGFP1-N1 vector (Clontech) and mouse Cathepsin S cDNA as template. The STOP codon was removed from the C-terminus by PCR and the sequence cloned in-frame into the vector. The transfected cells were seeded onto 6-well dishes or Matrigel-coated 12-well plates and cultured for 24 or 48 hrs. After incubation, the cells were either processed for flow cytometry analysis or fixed and processed with appropriate primary and fluorescent secondary antibodies and analyzed using confocal fluorescence microscopy.
Flow cytometry analysis revealed that nucleofection of LGACs with a control GFP plasmid (pmaxGFP) could be optimized to result in 47 ±2% transfection efficiency (n=4). The optimized conditions included using 6 x 106 cells per nucleofection reaction on day 1 of cell culture, with 3 µg plasmid DNA, on program Z-001. Confocal fluorescence microscopy and labeling of actin filaments and the mature secretory vesicle marker, Myosin 5c revealed that the normal morphology and secretory vesicle complement was maintained in transfected reconstituted acini after electroporation. No significant morphological changes were observed in the reconstituted acini, although additional single cells were detected in the culture. Electroporation on day 2 resulted in more reconstituted acini than electroporation on day 1 although transfection efficiency was slightly decreased. Additionally, transfection with a vesicle-targeted construct, cathepsin-GFP, resulted in overexpression of the protein and punctate, vesicle-associated labeling in basolateral regions, consistent with targeting to lysosomes.
Nucleofection offers an efficient way to deliver plasmid DNA to previously difficult-to-transfect primary differentiated epithelial cells. This technology offers a powerful, non-viral tool for further understanding of the lacrimal gland and related diseases.
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