Miniaturization of gene transfection assays in 384- and 1536-well microplates

Abstract

The miniaturization of gene transfer assays to either 384 or 1536-well plates greatly economizes the expense and allows much higher throughput when transfecting immortalized and primary cells compared to more conventional 96-well assays. To validate the approach, luciferase and GFP reporter gene transfer assays were developed to determine the influence of cell seeding number, transfection reagent to DNA ratios, transfection time, DNA dose, and luciferin dose on linearity and sensitivity. HepG2, CHO and 3T3 cells were transfected with polyethylenimine (PEI)-DNA in both 384 and 1536 well plates. The results established optimal transfection parameters in 384-well plates in an total assay volume of 35 μl and in 1536-well plates in a total volume of 8 μl. A luciferase assay performed in 384-well plates produced a Z' score of 0.53, making it acceptable for high throughput screening. Primary hepatocytes were harvested from mouse liver and transfected with PEI-DNA and calcium phosphate DNA nanoparticles in 384-well plates. Optimal transfection of primary hepatocytes was achieved on as few as 250 cells per well in 384-well plates, with CaPO4 proving to be 10-fold more potent than PEI.

Investigating Novel Syntheses of a Series of Unique Hybrid PLGA-Chitosan Polymers for Potential Therapeutic Delivery Applications

“…The discovery of effective therapeutic drugs is becoming increasingly difficult as seen by the drastic decline of new therapeutics accepted for public use each year. This is seen even with advances in structure activity relationship (SAR) studies [1], computer simulations of target structures (specific binding sequences and shape elucidation) [2], and high throughput screening methodology [3]. Novel surfaces and delivery nanosystems have taken the spotlight as the leading hope to advance new drugs from research into and beyond clinical studies by overcoming factors such as: lack of solubility, poor stability, poor biodistribution, immune response activation, off-target affects, and poor accumulation at the target site.…”

Cell Transfection-A Short Approach of DNA Mutagenesis

“…20 Liposomes formed by cationic lipids: The term "liposome" refers to lipid bi-layers that forms colloidal particles in an aqueous medium. 21,22 Artificial liposomes were being used to deliver DNA into cells, 23 the advances in liposomal vehicles were development of synthetic cationic lipids by Felgner et al 24 Liposome-mediated delivery offers advantages such as relatively high efficiency of gene transfers, ability to transfect certain cell types that are resistant to calcium phosphate or DEAE-dextran, in vitro and in vivo applications, successful delivery of DNA of all sizes of oligonucleotides at yeast artificial chromosomes, [24][25][26][27][28][29][30] delivery RNA 31,32 and protein. 33 While DNA will need to enter the nucleus, the cytoplasm is the site of action for RNA, protein or antisense oligonucleotides delivered via liposomes.…”

“Evolving nanoparticle gene delivery vectors for the liver: What has been learned in 30 years”

“…Due to the large particle size of the precipitates, calcium phosphate has remained mostly an in vitro transfection method. However, in vitro gene transfer efficiencies comparable to PEI are possible [102]. In addition, the particle size can be significantly decreased by controlling the mixing rate and ratio of calcium to phosphate ions.…”