Use of nucleofection to efficiently transfect primary rabbit lacrimal gland acinar cells

Abstract

Lacrimal gland acinar cells are an important cell type to study due to their role in production and release of tear proteins, a function essential for ocular surface integrity and normal visual acuity. However, mechanistic studies are often limited by problems with transfection using either plasmid DNA or siRNA. Although various gene delivery methods are available, many have been unproductive due to consistently low transfection efficiencies. We have developed a method using nucleofection that can result in 50% transfection efficiency and 60% knockdown efficiency for plasmid DNA and siRNA, respectively. These results are vastly improved relative to previous studies, demonstrating that nucleofection offers an efficient transfection technique for primary lacrimal gland acinar cells.

Fig. 1

Nucleofection of rabbit lacrimal gland acinar cells. Rabbit LGAC were seeded in 150-mm petri dishes at a density of 2 × 10⁶ cells/mL. On day 1 of culture, cells were nucleofected with 3 μg pmaxGFP. Nucleofected cells were analyzed for expression of GFP by fluorescence microscopy 24 h post-nucleofection (a) Bar 50 μm. Following microscopy imaging, the cells were prepared for FACS analysis and assayed by such (b). Overlay refers to the composite image including fluorescence and phase contrast. (For GFP, n = 4)

Fig. 2

Nucleofected LGAC are able to accurately localize a vesicular compartment protein. a Rabbit LGAC were seeded in 150-mm petri dishes at a density of 2 × 10⁶ cells/mL. On day 2 of culture, cells were or were not nucleofected with 3 μg of a Cathepsin-S-GFP plasmid and seeded on Matrigel-coated coverslips in 12-well plates as described in Materials and Methods. After 24 h, the cells were fixed and processed to fluorescently label Cathepsin (green) and actin filaments (red). A schematic is also included in which lumena are marked with an “L”. Note the expression of Cathepsin S-GFP localized to punctate organelles. Bar10 μm. b Control rabbit LGAC were fixed on day 3 and processed to fluorescently label actin (red) and nuclei (blue), which are basolaterally located. Bar 10 μm

Fig. 3

Transfection of rabbit lacrimal gland acinar cells. Rabbit LGAC were seeded in 150-mm petri dishes at a density of 2 × 10⁶ cells/mL. On day 2 of culture, cells were transfected with either Fugene 6 (a) or Lipofectamine 2000 (b) as described in “Materials and methods”. After 24 h, control and transfected cells were collected and prepared for analysis by flow cytometry. For comparison, HeLa cells were also transfected and analyzed in parallel (n = 3 for HeLa cells and n = 4 for LGAC)

Fig. 4

Effective gene expression knockdown. Rabbit LGAC were seeded in 150-mm petri dishes at a density of 2 × 10⁶ cells/mL. On day 2 of culture, cells were either transfected with 100 pmol CAR siRNA and 5 μL Lipofectamine, 2 μg CAR siRNA and 10 μL GeneSilencer, or nucleofected with 2 μM CAR siRNA or a non-targeting sequence, siControl. Afer 24 h, the total RNA from control and experimental samples was isolated, purified, and used for cDNA synthesis. Relative expression levels were determined by quantitative real-time PCR. Results were normalized to HPRT1 mRNA and are expressed relative to control, untreated cells (n = 4). *significant at p ≤ 0.05 relative to control. # significant at p ≤ 0.05 relative to Lipofectamine 2000

Fig. 5

Nucleofection does not affect cell viability. Rabbit LGAC were seeded in 150-mm petri dishes at a density of 2 × 10⁶ cells/mL. On day 2 of culture, cells were either mock-nucleofected or nucleofected with either 3 μg pmaxGFP or 2 μM siRNA. After 24 h, the cells were assayed using the LIVE/DEAD viability/cytotoxicity kit for mammalian cells (n = 4)