Technical Reference #1891

1891.

Gene delivery through the use of a hyaluronate-associated intracellularly degradable crosslinked polyethyleneimine Peisheng Xu; Griffin K. Quick; Yoon Yeo, Purdue University, Biomaterials, 30(1891), (2009)

Keywords:
Non-viral gene delivery; Crosslinked polyethyleneimine; Sodium hyaluronate; Extra- and intracellular stabilities; DNA unpacking

Materials & Methods:
For intracellular trafficking of the DNA–polymer complexes NIH/3T3 cells were plated in a 35-mm dish with a glass window (MatTek) at a density of 100000 cells ‘‘per dish’’ in 2 mL of culture medium. The DNA–polymer complexes were prepared with Cy3-labeled pBR322 plasmid. After overnight culture the medium was replaced with 2 mL of transfection medium containing the DNA–polymer complexes and the cells were incubated with the DNA–polymer complexes for 6 hours. The transfection medium was then replaced with the fresh complete medium. The cells were imaged with the confocal laser scanning microscope (Olympus X81) operated with Fluoview software (Olympus Japan). The DNA– polymer complexes were excited using a 488-nm laser and the emission signal was read from 560 to 630 nmand expressed in red. LysoTrackerÒwas added half an hour before the observation at the concentration of 150 nM and excited using a 488-nm laser. Its emission was read from 500 to 550 nm and expressed in green. The nuclei were stainedwith DRAQ5 (5 mM AXXORA LLC San Diego CA USA) and excited using a 633-nm laser and its emissionwas read from 650 to 750 nmand expressed in blue. Images were processed with the NIH ImageJ software (Version 1.36b National Institutes of Health USA). FRET imaging was performed to investigate the intracellular dissociation of the DPH complexes. The pBR322 plasmid was dual-labeled with Cy3 and Cy5 using the Label ITÒ Kit. According to manufacturer’s protocol the average density of fluorescent dyes is one dye molecule per 170 DNA base pairs. DPH complexes were prepared using the dual-labeled pBR322. NIH/3T3 cells were plated in a 35-mm dish with a glass window (MatTek) at a density of 100000 cells per dish and incubated for 24 hours. Transfection medium containing the DPH complexes was then added to cells at the concentration of 4 mg DNA per well. After 3 hours of incubation the transfection medium was removed by aspiration and the cells were washed twice with the fresh complete medium. The emission spectra of the DPH complexes were acquired with a confocal laser scanning microscope (Olympus X81) using the lambda function of the Fluoview software (Olympus Japan). Excited with a 543 nm laser the emission signals were collected stepwise from 550 to 750 nm at the step length of 5 nm and at the bandwidth of 5 nm.

Microscopic Technique
confocal laser scanning microscope

Cell Type(s)
NIH/3T3 cells