Cost-effective gene transfection by DNA compaction at pH 4.0 using acidified, long shelf-life polyethylenimine

Abstract

Introduction of genetic material into cells is an essential prerequisite for current research in molecular cell biology. Although transfection with commercially available reagents results in excellent gene expression, their high costs are obstacles to experimentation with a large number or large scales of transfection. The cationic polymer linear-polyethylenimine (MW 25,000) (PEI), one of the most cost-effective vehicles, facilitates DNA compaction by polyplex formation, which leads to efficient delivery of DNA into cells by endocytosis. However, the use of PEI is still limited because of substantial cytotoxicity and intolerable deterioration in transfection efficiency by its low stability. Here, we show that acidification of PEI is important for its transfection activity. Dissolving PEI powder in 0.2N HCl confers a long shelf-life for PEI storage at 4 and -80 degrees C, and the polyplex formation of plasmid DNA with PEI is optimized in lactate-buffered saline at pH 4.0. Furthermore, changing the culture medium at 8-12 h posttransfection can minimize the cytotoxicity of PEI without sacrificing the high transfection efficiency comparable to that of commercial reagents. The cost per test using acidified PEI is drastically reduced to approximately 1:10,000, compared with commercial reagents. Thus, we conclude that acidification of PEI satisfactorily accomplishes cost-effective, high-efficiency transfection.

Src Family Kinases Promote Silencing of ATR-Chk1 Signaling in Termination of DNA Damage Checkpoint

“…The expression of v-Src was induced with 1 g/ml doxycycline. Gene transfection was performed using acidified polyethylenimine (38,57) or Lipofectamine 2000 (Invitrogen). Parental HeLa S3 and HeLa S3 cells stably expressing NLS-Lyn or TR were cultured in Iscove's modified Dulbecco's medium supplemented with 5% bovine serum.…”

Src Acts as an Effector for Ku70-dependent Suppression of Apoptosis through Phosphorylation of Ku70 at Tyr-530

“…77; provided by T. Yamamoto) as described (18,22). The pcDNA4/TO vector (Invitrogen) encoding enhanced green fluorescent protein (EGFP) (pcDNA4-TO-EGFP) was constructed previously (78). Bax was generated by PCR from HeLa cell cDNAs with 5Ј-ACTGAATTCAGTGGAGGTATG-GACGGGTCCGGGGAGCAGCCCAG-3Ј (sense) and 5Ј-CAGTCTCGAGTCAGCCCATCTTCTTCCAGATGGT-GAGTGAGGC-3Ј (antisense), and the EcoRI-XhoI fragment of the PCR product was subcloned into the EcoRI-XhoI site of the pcDNA3/FLAG vector (5), resulting in the construction of FLAG-tagged Bax (FLAG-Bax).…”

“…HeLa S3/sh(Fyn) cells, HeLa S3/sh(Lyn) cells, and HeLa S3/sh(Fyn)ϩsh(Lyn) cells were generated by using pENTR4/H1/sh(Fyn) and/or pENTR4/H1/sh(Lyn) with a plasmid containing the hygromycin resistant gene as described previously (69). Cells seeded in a 35-mm (60-mm) culture dish were transiently transfected with 1 g (3 g) of plasmid DNA using 5 g (15 g) of linear polyethyleneimine (25 kDa) (Polyscience, Inc.) (78). To establish a stable Ku70-knockdown cell line (HeLa S3/shKu70), HeLa S3 cells were cotransfected with pENTR4/H1/shKu70 and a plasmid containing the hygromycin resistant gene and were selected in 250 g/ml hygromycin.…”

Poly-ion Complex of Chondroitin Sulfate and Spermine and Its Effect on Oral Chondroitin Sulfate Bioavailability

“…[21][22][23][24][25] Based on these insights, SPM and polyamine analogs such as poly (L-lysine), polyethylenimine, etc., have been examined for the construction of nanoparticles containing small interfering RNA (siRNA) or plasmid DNA for delivering genes to specific target organs or cultured cells. [26][27][28][29][30][31] Interestingly, poly-ion complex (PIC) formation through the electrostatic interaction between polylysine and CS, has been reported. 32) This report inspired us to attempt to prepare PICs by mixing CS and natural polyamines in aqueous solutions and investigate their effect on CS oral absorption.…”