October 1, 2010 (Vol. 30, No. 17)
Joshua Bryson, Ph.D.
Biodegradable Polymer-Based DNA Carrier Designed to Facilitate Process in Mammalian Cells
Delivering nucleic acids such as siRNA, mRNA, antisense agents, transcription factor decoys, and plasmid DNA offers a vast untapped potential for studying many new genetic factors for their relationship with various devastating human diseases. The barrier to the use of nucleic acids for the development of customized and specific treatments is safe, efficacious delivery of DNA.
Delivery method choice is an important factor that contributes to total transgene expression, level of throughput, and cell viability, which is especially critical in primary cells that are costly and difficult to obtain. Indeed, high cell viability becomes even more essential when transfection is carried out, in sensitive cell types, in vivo, or in ex vivo implanted cells.
Traditional means of carrying DNA into cells rely on cell membrane disruption such as electroporation or infection with viruses. Both of these have their own positive attributes and drawbacks but are generally not conducive to rapid high-throughput screening of multiple DNA constructs.
Other approaches relying on chemical agents such as lipids and polymers provide an improved means for high-throughput DNA screening in immortal cell lines, but few are designed with primary and sensitive cell types in mind.
Techulon has launched a new polymer-based transfection reagent, Glycofect™, which is designed specifically for DNA transfection in primary cells. Glycofect is a carbohydrate-containing polyamidoamine that binds with DNA of various sizes and forms positively charged nanoparticles called polyplexes. Glycofect has water-labile bonds that biodegrade during cellular uptake, enabling the release of DNA in a cell’s perinuclear region for maximum gene expression.
Studies have indicated that Glycofect is preferentially uptaken by cells via a caveolae-mediated endocytotic route, which contributes to the high nuclear delivery. Glycofect has been tested with various reporter plasmids and labeled oligo DNA and is proving to be an effective tool to the cell therapy and molecular biology communities.
In this article, we employ various means of testing gene expression and cell viability for Glycofect and several other commercially available transfection reagents in primary neonatal human dermal fibroblasts (HDFn), a cell of high therapeutic importance for its use in skin grafts and for making induced pluripotent stem cells.
We compare gene expression of various reagents based on transgene luciferase activity. Further, we quantify gene expression on a per cell basis with enhanced green florescent protein expression, using both florescent microscopy and flow cytometry to quantify results. Finally, to quantify the amount of healthy cells remaining after transfection, we test the viability of these cells using both the MTT and BCA assays.
Luciferase and BCA Assay. Prior to transfection, HDFn cells (Invitrogen) were plated on 24-well plates at a density of 100,000 cells per well, approximately 70% confluency. Cells were incubated in Medium 106, supplemented with 2% FBS, hydrocortisone (1 µg/mL), human epidermal growth factor (1 ng/mL), basic fibroblast growth factor (3 ng/mL), and heparin (10 µg/mL), for 24 hours at 37ºC in a 5% CO2 environment.
Cells were starved of sera 30 minutes prior to transfection. Transfection reagents (two leading lipid reagents and Glycofect) were formulated with pDNA based upon their recommended protocols. Solutions of transfection reagent-pDNA (gWiz-Luciferase, Aldevron) complexes for each transfection reagent were added in triplicate to corresponding wells (1 µg pDNA per well). Plates were briefly swirled and incubated for four hours, after which more media (500 µL) was added to each well. Cells were incubated for an additional 20 hours, followed by a media change and 24 hours of additional incubation time. Media was evacuated from wells and cells were lysed in 100 µL Cell Lysis Buffer (Promega).
Cell lysates were deposited on 96-well plates and analyzed for luciferase activity on a luminometer plate reader (Tecan GENios Pro). Protein lysates were stained using a BCA staining procedure, and their concentration was determined with absorbance measurements at 750 nm. Cell viability was determined by sample absorbance relative to the “cells only” control.
MTT Assay. Cells were prepared and transfected using the same methodology as for the luciferase and BCA assays. However, at the 47 hour time point media was evacuated from each well and replaced with media containing 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, [MTT] = 0.5 mg/mL). Cells were incubated for an additional 1 hour then washed with PBS and lysed in 250 µL DMSO. Sample cell lysates were analyzed via absorbance vs. cells only lysate’s absorbance to determine cell viability.
GFP Analysis via Flow Cytometry. Cells were plated on 6-wells and polyplexes were formed using the same methods reported using a plasmid-encoding enhanced green fluorescent protein (EGFP-C1, 5µg total per well). Transfection and media change conditions are consistent with those reported above. After 48 hours cells were trypsinized, washed with PBS twice, and suspended in 500 µL PBS. Flow cytometry analysis of each sample provided mean fluorescence intensity as well as the percentage of cells positive for EGFP.
GFP Analysis via Fluorescent Microscopy. Cells were plated using the same methods performed for the flow cytometry experiment. Transfection and media change conditions are also consistent with those reported above. After 48 hours, cells were washed with PBS twice and fixed in a 4% para-formaldehyde PBS buffer solution. Cells were imaged on a Nikon TE2000U fluorescent microscope for both differential interference contrast images and for GFP florescence (ex. 488 nm, em. 509 nm).
Luciferase Expression and Toxicity Profiles. In HDFn cells, only lipid reagent A and Glycofect elicited a positive response for luciferase expression (Figure 1A) compared with controls. Both lipid B and DNA only showed no transfection. Although some toxic response was observed for each transfection reagent used, minimal toxicity, confirmed by both MTT and BCA assays (Figure 1B), was observed in the Glycofect wells after 48 hours.
EGFP Expression in HDFn. Total gene expression per cell was carried out via flow cytometry using EGFP. Flow cytometry indicated that both lipid reagent A and Glycofect enabled the expression of EGFP beyond the positive threshold gate in over 50% of the cell population. The sample transfected with naked DNA showed no transfection, and lipid reagent B showed low expression (13.3%). Although a higher percentage of cells are expressing with lipid reagent A (58.4%), cells show greater signal intensity in the Glycofect sample (Figure 2). Micrographs of these trials further verify this trend (Figure 3).
The Glycofect reagent has demonstrated improved DNA transfection in primary cells by showing high gene expression in a high percentage of cells, while maintaining low toxicity.
Reporter gene data shows that Glycofect is an effective transfection reagent in human dermal fibroblast cells and has the potential to be used in therapies requiring transient genetic alteration of this cell type.