PEI transfection patents to expire

July 7, 2015 marked the 20-year anniversary of the filing dates of both the U.S. patent and European patent limiting the use of PolyEthylenImine (PEI) as a transfection reagent. Coincidentally, both U.S. and European patents generally have a term of 20 years from the filing date.

Many academic researchers have been ignoring these patents and/or have been sharing protocols online and publishing articles explaining how cost-effective and simple PEI-mediated transfection can be. For example, the most commonly used PEI, catalog nr. 07923966-2 (Polysciences), is a linear form with molecular weight of 25,000 Da. The 2 gram bottle of PEI powder can be used to make a few liters of transfection reagent, so depending on which protocol is used the cost can be about 0.01% that of commercially-available transfection reagents.

PEI structure. Image source: https://en.wikipedia.org/wiki/Polyethylenimine

PEI structure. Source: Wikipedia.

PEI is a simple polymer with an amine group and two carbon spacer that is thought to bind to DNA to produce positively charged particles that can enter the cell.

Some of the negative aspects of PEI compared to more sophisticated transfection reagents are:

  1. PEI tends to be rather cytotoxic. While PolyJet™ DNA In Vitro Transfection Reagent  is composed of proprietary bio-degradable polymers designed to greatly reduce cytotoxicity, standard PEI has been shown to induce apoptosis in a variety of human cells.
  2. PEI is difficult to dissolve. Linear PEI is solid at room temperature and somewhat soluble in hot water and low pH. A typical protocol might involve dissolving 8mg of PEI in 25mL of water, a process that may take many hours or days and/or result in a non-uniform solution that cannot be sterile filtered.

To solve this problem, Polysciences has released the a much more soluble hydrochloride salt form (catalog nr 07924765-2), called Polyethylenimine “Max”, (Mw 40,000) – High Potency Linear PEI. This MW 40,000 form corresponds to the MW 25,000 polymer length in free base form, with the salt accounting for the molecular weight difference.

PEI max structure.

PEI max structure.

Looking through the literature one might reach the conclusion that PEI is a universal transfection reagent suitable for transfecting cultured cells or use in vivo.

Here’s a summary of some of the earlier scientific articles using PEI for various transfection applications:

Nucleic Acid Cell Type Publication
68-mers Hepatocytes ref
antisense oligos Neurons ref
BAC DNA mouse (in vivo) ref
DNA 293E ref
DNA Adult neural stem cells ref
DNA Brain derived cells ref
DNA chicken (in vivo) ref
DNA CHO cells ref
DNA Cos-1 ref
DNA Cos-7 (nuclei) ref
DNA Embryonic neurons (in vivo) ref
DNA Fetal mouse liver ref
DNA HeLa cells ref
DNA Human monocytes ref
DNA HUVEC ref
DNA L929 cells ref
DNA LNCaP cells ref
DNA Mouse brain ref
DNA Mouse lung ref
DNA Mouse lung ref
DNA Murine adult neural stem cells ref
DNA Ovarian carcinoma cells ref
DNA Postmitotic neurons ref
DNA Pseudocystic tumor cells ref
DNA Rat (in vivo) ref
DNA Rat brain ref
DNA Rat fetal hypothalamic cells ref
DNA Rat kidney ref
DNA Rat kidney ref
DNA WERI-Rb1 retinoblastoma ref
DNA Xenopus tadpole brain ref
dsRNA and siRNA Snail, Biomphalaria glabrata ref
modified siRNA Murine melanoma cells ref
siRNA in vivo ref
siRNA Pancreatic cancer cells ref
YAC HT1080 cells ref

 

 

 

 

 

 

 

 

 

In addition to use as a direct transfection reagent, PEI is used for a variety of “combination” gene delivery methods. Adenofection for example makes use of PEI to permit delivery of large plasmids/BAC/YAC into cells by non-covalently coupling of the DNA to an adenovirus. A variety of other approaches make use of modified (e.g. PEGylated) PEI for gene delivery or have targeted PEI to specific tissues with antibodies or proteins.

In the list of applications, mRNA delivery into cells seems conspicuously absent. Indeed at least one paper indicates that PEI-mediated transfection of mRNA is very inefficient. For mRNA transfection, researchers have better success with the Stemfect™ RNA Transfection Kit or the mRNA-In Transfection Reagent.

Transfection reagents by tebu-bio

HA tagged beta-tubulin cDNA was delivered into CHO cells. HA-beta-tubulin (Green) – Endogenous alpha-tubulin (Red).

 

Leave your comment below to share your experience with PEI!

 

 

 

 

 

 

 

 

 

 

 

 

 

HD-LCI video of HUVEC Transfection with Cytofect Kit

See human cells glow with green fluorescent protein in this time-lapse video. These images were captured over 20 hours via Lumascope 620. [Read more…]

Alternatives to the “mega-brands” of molecular biology

If you don’t buy luxury brand clothes, why are you still buying luxury brand molecular biology kits?

Let me guess… you might use those blue and red boxed Qiagen® kits for DNA and RNA purification, maybe TRIzol® if you’re a real rebel. You probably also use a SuperScript® RT Kit for reverse transcription and you can’t keep track of which company most recently bought Invitrogen® to control that logo that you’re loyal to.

Maybe it used to be the case that these were the only companies making these kits, so you really had no choice. Now, many of these products are essentially commodities, but you’re still used to purchasing the “first to market” brand because it works and it would probably cause trouble for your lab to change brands. The perceived “switching costs” are large part of the reason for this persistent brand loyalty, and part of the reason why ThermoFisher was willing to pay $13.6 billion for the Life Technologies brand in 2013. There is an entire arm of corporate finance devoted to assigning the value to a brand. Essentially, it tries to estimate how much more customers are willing to pay for a commodity item just because of the company logo on the side of the package.

Here, we examine some alternatives to 2 of the biggest molecular biology mega-brands that seem to be able to keep their loyal customers by placing their familiar brand name on a relatively low value item:

1 – Alternatives to Lipofectamine® Reagents

There’s the one with the blue cap, the one with the red cap, and the one with the green cap, and most people don’t really understand the difference, but cell biologists seem to love these transfection reagents and are willing to pay a heavy premium for them.
Compared to the prices announced in 2015 on the Life Tech website for the 1.5 mL size Lipofectamine® 2000, similar products from competitors are available for about 1/4 the price:

LipoD293 DNA (Ver. II)  1 ml

– Enhanced conjugate with liposome for maximal efficiency
– Best for lentivirus, antibody and protein production

GenMute™ siRNA  1 ml

– Exceptionally good for siRNA transfection
– Excellent for DNA/siRNA co-transfection

LipoJet™ DNA  1 ml

– Fluorinated cationic lipids
– Best transfection efficiency with least toxicity

Validation data for Lipojet™, for example, suggest that it is superior to many other commercial transfection reagents including “L2K”::

Lipojet Multiple Cell Line Data

Lipojet Multiple Cell Line Data

LipoJet_L2K_PolyJet_293T

Given the vast selection of transfection reagents available today, the best option is usually to contact a technical specialist (at tebu-bio for example) for advice on the most suitable ones for your research.

2 – Alternatives to Qiagen® and TRIzol®

Whether you’re using the DNeasy® and RNeasy® kits or TRIzol® products for DNA and RNA extraction, chances are you just keep buying the same kit you learned to use when you were a student. Sure, there are companies making less expensive column-based kits, and there are even protocols online explaining how to regenerate the used columns and make your own buffers. You can also find generic RNAzol RT RNA Isolation Reagent. 

The advances we really like, however, are the purification systems that work without columns and without dangerous solvents. Here are a few of our favourites:

QuickExtract™ DNA Extraction Solution The fastest, least expensive option when you just need to purify DNA for PCR.

QuickExtract™ RNA Extraction Kit  Available in two sizes: the extremely competitively priced 5mL kit contains sufficient material to perform 50 RNA extractions, while the 50mL kit contains sufficient material to perform 500 RNA extractions.

MasterPure™ DNA and RNA Purification Kits  These are for the times when you need the highest quality nucleic acid for next generation sequencing, qPCR, or PCR. Produced by the RNA biology experts at Epicentre (an Illumina company), MasterPure™ Kits are available for nearly every application and source including mammalian cells, yeast, blood, Gram Positive bacteria, and Plant Leaves.

As is the case with transfection reagents, choosing a new nucleic acid purification kit is not always easy, so feel free to contact a technical specialist at tebu-bio for advice.

To get in touch, just leave your comments or questions below.

 

DNeasy® and RNeasy®, and Qiagen® are registered trademarks of the Qiagen group. TRIzol® is a registered trademark of Molecular Research Center, Inc. Invitrogen® and SuperScript® are registered trademarks of Invitrogen. Lipofectamine® is a registered trademark of Life Technologies, Inc. 

mRNA delivery tools

Direct delivery of RNA sequences to a cell circumvents many drawbacks inherent to plasmid or viral DNA. This innocuous strategy reveals being as efficient as viruses when it comes to conveying and expressing nucleic acid sequences in non-dividing cells, for it does not rely on nuclear entry, precluding any mutagenic events by the same token.

Once efficiently engineered to escape their automatic and swift destruction in most biological environments, RNA molecules become remarkably stable and turn out to be extremely reliable for in vivo applications. In line with our Nucleic acid delivery tools presentation series, let’s focus here on RNA delivery to the cell. [Read more…]

Your neurons will forget Lipofectamine!

Mature cultures of neurons and other neural cells are extremely valuable for in vitro neurotoxicity studies and screening for agents that can slow, stop, or even reverse the course of neurodegenerative diseases.

The transfection of nucleic acids into neurons is essential for studying many aspects of neurobiology. However, neurons are among the most difficult cell types to transfect. They are very sensitive to culture conditions, presenting a particular challenge with regards to efficiencies. In addition to yielding low efficiencies, currently available cationic lipid reagents are often toxic to the cells, compromising post-transfection experimental results. While some viral mediated gene delivery systems have been shown to produce high efficiencies, they are very labor intensive and inconvenient for most researchers, along with the inherent danger and risk of provoking an immune response in the cell and/or interfering with the host genome. So what other solutions exist? This recently introduced alternative is well worth discovering… [Read more…]

Tools for gene delivery

One of the most tantalizing abilities of human nature when facing a “what’s that for?” question, is to interfere in a system and check the downstream effects of this change. This empirical approach has set the basis of our global understanding of nature along history. Research is no different: functional study of biological systems is based on over-stimulating or inhibiting components and observing downstream consequences.

For any cell biologist, the target to be altered is the cell. The way to do this is by using molecules able to get through the membrane and produce an alteration (inhibition or stimulation) of the biochemistry inside. However, the plasmic membrane is designed to protect the cell from external “non-authorized intrusions” with very effective systems for this task. Therefore, how to efficiently deliver exogenous nucleic acids inside the cell?

[Read more…]