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Optimization is Essential to Increase Transfection Efficiency and Cell Viability in Primary and Difficult‐to‐Transfect cell lines
Author(s) -
Jordan Elizabeth T.,
Collins Michelle,
Terefe Joseph,
Pineda Maxinne,
Ugozzoli Luis,
Rubio Teresa
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.1_supplement.1003.18
Subject(s) - electroporation , transfection , jurkat cells , microbiology and biotechnology , viability assay , cell culture , biology , cell , small interfering rna , chemistry , t cell , immunology , gene , biochemistry , genetics , immune system
Electroporation is a valuable tool for nucleic acid delivery because it can be used for a wide variety of cell types. Many scientists are shifting towards the use of cell types of increasing scientific interest, including lineage‐specific cell types, bone marrow cells and stem cells, but these cells can be difficult to transfect. The ability to transfect these cell types with nucleic acid molecules of interest at a relatively high efficiency while maintaining cell viability would offer a new tool towards the elucidation of the pathway(s) in which a gene product is involved. In this poster, we present data demonstrating that optimization of electroporation parameters is essential to deliver molecules in a highly efficient manner. We display results of transfection of several difficult to transfect cell types, including Human Primary Fibroblasts, HUVEC, Jurkat and (mouse) Neuro‐2A neural cells with plasmid DNAs and siRNAs. We used multiple detection methods to determine transfection efficiency, including flow cytometry, real‐time PCR and the luciferase assay. Gene Pulser electroporation buffer and square or exponential wave pulses were used to convey molecules of interest into the cells. Optimization of electroporation parameters was carried out using the high throughput Gene Pulser MXcell electroporation system. Just 4 hours post electroporation with a GAPDH siRNA, Jurkat cells expressed 88% less GAPDH mRNA than controls transfected with scramble siRNA. The same degree of silencing persisted at 24 and 48 hours post electroporation. Transfection of cells with fluorescently labeled siRNA resulted in 75% transfection efficiency for Neuro‐2A, 93% for HPF and 98% for HUVEC cells, as analyzed by flow cytometry. We provide optimized electroporation conditions for these and other difficult to transfect cells.