Ingenio® Solution Efficiently Delivers siRNA, miRNA and Viral RNA

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Potent siRNA Knockdown of Transiently Expressed Firefly Luciferase Using the Ingenio

Ingenio® Kits Deliver siRNA for Knockdown Using Different Electroporation Instruments. siRNA and plasmid DNA were co-electroporated with the indicated cell lines with the Ingenio® electroporation solution in 0.2 cm cuvettes using either the Gene Pulser (Bio-Rad®) or the Amaxa® Nucleofector® II (Lonza). 10µg/ml of plasmid encoding Firefly luciferase was co-electroporated with 250nM of either non-targeting siRNA control (Dharmacon Non-Targeting siRNA #1) or GL3 siRNA into Jurkat E6-1, HeLa and CHO-K1 cells. Twenty-four hours post electroporation, cells were harvested and assayed for luciferase activity. Data from independent experiments performed on different days were averaged then scaled to non-targeting siRNA control and are represented as a percentage of the control.

Pulse conditions used:

(1) Amaxa® Nucleofector® program:
a. Jurkat E6-1: X-001 with 10 x 106 cells/ml
b. HeLa: I-013 with 3 x 106 cells/ml
c. CHO-K1: U-023 with 5 x 106 cells/ml

(2) Bio-Rad® Gene Pulser Exponential Decay setting:
a. Jurkat: 150V, 950µF with 10 x 106 cells/ml
b. HeLa : 130V, 950µF with 3 x 106 cells/ml
c. CHO-K1: 150V, 900µF with 5 x 106 cells/ml

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CRISPR RNP Delivery with Ingenio Electroporation Solution Targeting PPIB

CRISPR RNP Delivery with Ingenio® Electroporation Solution. K562 and Jurkat cells were electroporated with a Cas9 protein/gRNA, ribonucleoprotein (RNP) complex, composed of 750 nM Cas9 protein (EnGen® Cas9 NLS, NEB) and 1500 nM pre-complexed two-part gRNA (IDT) targeting PPIB using the Ingenio® Electroporation Solution and a Gene Pulser Xcell™ Eukaryotic System. Exponential pulse conditions of 130V, 950 µF for K562 and 150V, 950 µF for Jurkat cells were applied to triplicate 0.2 cm cuvettes, 100 µl volume, 10 x 106 cells/ml +/- RNP complex. A T7E1 mismatch assay was used to measure cleavage efficiency at 48 hours post-transfection. Non-specific bands (NSP) were observed in the negative control of both cell lines. Cleavage efficiency was calculated based on the ratio of cleaved band intensities to the sum of cleaved and uncleaved band intensities minus the average signal of the non-specific band(s) in negative control lanes.

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High Efficiency Plasmid DNA Electroporation of Human Induced Pluripotent Stem (iPS) Cells Using Ingenio

High Efficiency Plasmid DNA Electroporation of Human Induced Pluripotent Stem (iPS) Cells Using Ingenio®. The Ingenio® Electroporation Kit was used to transfect 2 x 106 iPS cells on the Amaxa® Nucleofector® II Device. Cells were electroporated with 8 µg ZsGreen expressing plasmid (Clontech) in 100 µl and plated in 6-well plates at 0.33 x 106 cells/well. Cells were visualized 24 hours post-transfection and imaged under 4X objective with an Olympus IX71® Inverted Microscope. Images are (A) phase contrast, and (B) green fluorescence. Cells were also assayed 24 hours post-transfection on an Accuri® Cytometer. The histogram (C) shows unelectroporated cells (black line) compared to cells electroporated with plasmid using the Ingenio® Electroporation Kit (green line). See more information on stem cell applications.

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