Jing Li1, Samuel T Crowley1, Jason Duskey1, Sanjib Khargharia1, Meng Wu2, Kevin G Rice3. 1. Division of Medicinal and Natural Products Chemistry, College of Pharmacy, University of Iowa, Iowa City, IA 52242, USA. 2. Division of Medicinal and Natural Products Chemistry, College of Pharmacy, University of Iowa, Iowa City, IA 52242, USA; Department of Biochemistry, University of Iowa, Iowa City, IA 52242, USA; University of Iowa High Throughput Screening Facility, University of Iowa, Iowa City, IA 52242, USA. 3. Division of Medicinal and Natural Products Chemistry, College of Pharmacy, University of Iowa, Iowa City, IA 52242, USA. Electronic address: kevin-rice@uiowa.edu.
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 with more conventional 96-well assays. To validate the approach, luciferase and green fluorescent protein (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 NIH 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 a total assay volume of 35μl and in 1536-well plates in a total assay volume of 8μl. 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 250cellsperwell in 384-well plates, with CaPO4 proving to be 10-fold more potent than PEI.
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 with more conventional 96-well assays. To validate the approach, luciferase and green fluorescent protein (GFP) reporter gene transfer assays were developed to determine the influence of cell seeding number, transfection reagent to DNA n class="Species">ratios, tranpan>sfection time, DNA dose, anpan>d pan> class="Chemical">luciferin dose on linearity and sensitivity. HepG2, CHO, and NIH 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 a total assay volume of 35μl and in 1536-well plates in a total assay volume of 8μl. 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 250cellsperwell in 384-well plates, with CaPO4 proving to be 10-fold more potent than PEI.
Authors: I Kariv; H Cao; P D Marvil; E V Bobkova; Y E Bukhtiyarov; Y P Yan; U Patel; L Coudurier; T D Chung; K R Oldenburg Journal: J Biomol Screen Date: 2001-08
Authors: Henry K Bayele; T Sakthivel; Martin O'Donell; K John Pasi; Andrew F Wilderspin; Christine A Lee; Istvan Toth; Alexander T Florence Journal: J Pharm Sci Date: 2005-02 Impact factor: 3.534
Authors: Glòria Sanclimens; Hong Shen; Ernest Giralt; Fernando Albericio; Mark William Saltzman; Miriam Royo Journal: Biopolymers Date: 2005 Impact factor: 2.505
Authors: Mina Edwards; Siew Cheng Wong; Roongsiri Chotpadiwetkul; Despina Smirlis; lan R Phillips; Elizabeth A Shephard Journal: Methods Mol Biol Date: 2006
Authors: Jason Thomas Duskey; Cecilia Baraldi; Maria Cristina Gamberini; Ilaria Ottonelli; Federica Da Ros; Giovanni Tosi; Flavio Forni; Maria Angela Vandelli; Barbara Ruozi Journal: Polymers (Basel) Date: 2020-04-04 Impact factor: 4.329