Journal article
Gene transfection of primary mouse hepatocytes in 384-well plates
Analytical biochemistry, Vol.644, pp.113911-113911
05/01/2022
DOI: 10.1016/j.ab.2020.113911
PMCID: PMC7936984
PMID: 32910973
Abstract
We report the development of an improved in vitro transfection assay to test the efficiency of non-viral vector DNA nanoparticle transfection of primary hepatocytes. The protocol describes the isolation of viable hepatocytes from a mouse by collagenous perfusion. Primary mouse hepatocytes are plated in 384-well plates and cultured for 24 h prior to transfection with polyethylenimine (PEI) or peptide DNA nanoparticles. Luciferase expression is measured after 24 h following the addition of ONE-Glo substrate. The gene transfer assay for primary hepatocytes was optimized for cell plating number, DNA dose, and PEI to DNA ratio. The assay was applied to compare the expression mediated by mRNA relative to two plasmids possessing different promoters. The reported assay provides reliable in vitro expression results that allow direct comparison of the efficiency of different non-viral gene delivery vectors.
Details
- Title: Subtitle
- Gene transfection of primary mouse hepatocytes in 384-well plates
- Creators
- Raghu Ramanathan - University of IowaNathan A. Delvaux - University of IowaKevin G. Rice - Univ Iowa, Coll Pharm, Div Med & Nat Prod Chem, Iowa City, IA 52242 USA
- Resource Type
- Journal article
- Publication Details
- Analytical biochemistry, Vol.644, pp.113911-113911
- DOI
- 10.1016/j.ab.2020.113911
- PMID
- 32910973
- PMCID
- PMC7936984
- NLM abbreviation
- Anal Biochem
- ISSN
- 0003-2697
- eISSN
- 1096-0309
- Publisher
- Elsevier
- Number of pages
- 5
- Grant note
- GM117785; T32 GM00865 / NIH; United States Department of Health & Human Services; National Institutes of Health (NIH) - USA
- Language
- English
- Date published
- 05/01/2022
- Academic Unit
- Pharmaceutical Sciences and Experimental Therapeutics; Craniofacial Anomalies Research Center; Medicinal and Natural Products Chemistry
- Record Identifier
- 9984365886702771
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