The Kinetics of Polyethylenimine-Mediated Transfection in Suspension Cultures of Chinese Hamster Ovary Cells

Abstract

The kinetics of polyethylenimine (PEI)-mediated gene transfer at early times after transfection of Chinese hamster ovary (CHO) cell in suspension were investigated using a novel in vitro assay. Addition of an excess of competitor DNA to the culture medium at various times after the initiation of transfection inhibited further cellular uptake of PEI-DNA particles. Using this approach, a constant rate of particle uptake was observed during the first 60 min of transfection at a PEI:DNA ratio of 2:1 (w/w) and a cell density of 2 x 10(6) cells/ml under serum-free conditions. The uptake rate declined considerably during the next 2 h of transfection. Both the rate and the level of PEI-DNA uptake in serum-free minimal medium were found to be dependent on the PEI-DNA ratio, the cell density at the time of transfection, and the extent of particle aggregation. These studies of the early phase of PEI-mediated transfection are expected to lead to further opportunities for optimization of gene transfer to suspension cultures of mammalian cells for the purpose of large-scale transient recombinant protein production.

Interactome analysis of AMP-activated protein kinase (AMPK)-α1 and -β1 in INS-1 pancreatic beta-cells by affinity purification-mass spectrometry

“…Alternatively, we overexpressed N-terminally 6-myc-tagged rat AMPK-α1 and AMPK-β1 in HEK293T cells and immobilized them to Dynabeads (Figure 1A, Bait production). Briefly, to express the recombinant proteins, full-length rat AMPK-α1 and -β1 cDNA was subcloned into modified pcDNA3.1 and transfected into HEK293T cells by PEI method2324.…”

“…The plasmids were transfected into HEK293T cells by PEI method2324. Prior to transfection, 12 μg of plasmid in 2 ml serum-free media were mixed with 36 μl PEI solution (1 μg/μl) and incubated for 15 min at room temperature.…”

Efficient mAb production in CHO cells incorporating PEI‐mediated transfection, mild hypothermia and the co‐expression of XBP‐1

“…Careful examination of the literature highlights that many variables contribute to the successful formation of PEI‐plasmid DNA complexes 12, 19, 29, 55, 56. In this work a multi‐parametric study was performed to improve PEI‐mediated transfections with the Epi ‐CHO expression system by examining cell seeding density, DNA:PEI ratios and DNA concentrations.…”

Use of an anti‐apoptotic CHO cell line for transient gene expression

“…Varying the molar ratio of PEI (N) to DNA (P) can significantly impact transfection yields . To identify conditions that provide the highest transient transfection productivity for the DKO and CHO‐K1 cell lines, full factorial experiments were conducted using different combinations of DNA and PEI volumes.…”