Dear Ravi,

There’re many factors that may influence the transfection efficiency, such as cell surface receptors, immune responses and so on. To establish stable cell lines, lentivirus is really an efficient way to for gene ORF expression, shRNA/miRNA and CRISPR/Cas9. However, despite mediating efficient transfection in both dividing and non-dividing cells, for some non-dividing primary cells, it is still difficult for lentivirus to transfect genes. Then, high-titer adenovirus to infect these cells like BMSC is recommended. While for in vivo experiment, the AAV viruses work best.

Genemedi got a rich experience in virus production, you could find more information and experience on https://www.genemedi.net/i/lentivirus-packaging; https://www.genemedi.net/i/adenovirus-packaging; https://www.genemedi.net/i/aav-packaging

Hope this may help you.

Hi,

While I haven't gone through any article based on your query, its one of the most common questions we come across. According to what I understand, transfection that is when you are trying to send your DNA of interest in..you essentially are looking for best membrane poration or entry through different methods.

Every cell line and primary cell line, a suspension or adherent cell line will have a different property and composition of its membrane. The agents which we use for the delivery pf desired gene of interest is either viral, physical or chemical right. So based on the charge imparted by the plasma membrane of your cell type and the chemical interaction of say a poly cationic polymer or lipid based reagent would interact and determine the efficiency of entry as well as the no, of cells they can enter as a complex.

When we say hard to transfect, it would mean that the membrane is difficult to porate or pass through. the bulky component like a DNA-lipid complex or Ca2+ based cations are not good enough to form an entry or there is a steric effect. In those cases viral based entries are effective as there is a fusion mechanism and it would work only better if you have exposed receptors.

So for me, the orientation of a cell membrane, thickness, charge on the membrane due to ration of lipid and cholesterol etc make a cell hard or easy to transfect.

Unfortunately, there is a huge variability in cell transfection efficiency, for all the reasons listed above, and that's in part why a lot of experiments are carried out in cells like HeLa or HEK293, eventhough those are not the best cells to model human disease... If you are trying to transfect lymphoids cells or primary, you can try some of the Amaxa transfection reagents, which are designed for specific cell types. Otherwise if you are ok with stable cell lines, the best way to go is retro or lentivirus. Best of luck.

Well, to add to Kalpita's comments, there is at least one other factor. Getting your DNA (or RNA) in the cell is not a natural process (at least at these quantities and conditions as in case of transfection). Therefore, transfection is inherently toxic (there are ways of avoiding this, but still, toxic). Easy to transfect cells may just more easily "ignore" transfection, or be more resistant to toxic effects,

Then there is the factor of suspension cells (hard to transfect) vs attached cells (easyer to transfect). Clearly, the local concentration of the transfection media plays important role; lipid nanoparticles with the DNA sediment to the bottom of the dish, increasing the (local) concentration of your transfection DNA.

Thank you for the responses. I do have the experience with various methods for transfections described by all of you, and had success most of the time. However what I am trying to understand "Why"such differences exists. How the 'easy to' and 'hard to' transfect cells are different. It looks to me thatthere is no simple answer for this question, otherwise the biotech companies would have figured it out. I am after an answer for this question because I am trying to understand what would be biological significance for such differences, there must a reason why certain cells take up transfection complex easily than certain others, in general transformed epithelial cells come under easy to transfect category, while primary cells, fibroblasts, lymphoid cells etc. are not so flexible, is this due to cancerous behavior, does that mean gene delivery to cancer cells should be easier and may spare normal cells or cancer cells could pick up viruses and tranform further and such questions.

Thank You

While it is difficult but not impossible. I had a chance to transfect airway smooth cells isolated from human and guineapigs. If you need further help contact me on [email protected]

Non-adherent cells are hard to transfect because they do not possess cell surface heparan sulfate proteoglycans. These are responsible for adhesion of cells to the extra-cellular matrix.

Cationic species can enter the cells through the membrane because cells are capacitatively charged (negative charge). Too high positive charge can however poke permanent holes in the cells thereby causing cell lysis. Certain viruses that infect non-adherent cells do so by using cell-penetrating peptides, which is simply a sequence of positively charged amino acids. Electrical pulse can also open transient pores in the cell membrane, but too high of the voltage amplitude can again poke permanent holes causing cell lysis.

Hi,

In order to offer better chances of success, Magnetofection is currently a simple method  particularly suitable for primary and hard to transfect cells.

In brief, Magnetofection associates magnetic nanoparticles bearing properties to bind nucleic acids with a strong magnetic field in order to attract and concentrate vectors onto the cell surface. You can refer to this web page  https://en.wikipedia.org/wiki/Magnetofection

Do not hesitate to contact me for any request.

For HEK 293, specifically, the cell line was initially transformed with adenoviral DNA, making it overall easier for the cell to both accept nucleic acids and translate them into proteins. Other cell types, like stem cells, are generally not ready to translate any given sequence, and require a lot of attention to make sure that they are in the right environment for the type of transfection being executed. There's two main parts here as to why a transfection might be too difficult with given cells:

1) Getting your complexes across the cell membrane, which can be toxic to the cells. There are many solutions to this problem (see https://altogen.com/product/hek-293-transfection-reagent-epithelial-kidney-cells/), and usually they involve having the cell accept the complexes without having to destroy the entire cell membrane.

2) Getting your nucleic acids/proteins expressed. Cells may not be amenable to transfection because they in general don't express foreign sequences. Hence you have to make sure your promoter regions and other associated signals are well-designed and tailored to the cell line you are using.

Hope that helps!

Dear Ravi,

There’re many factors that may influence the transfection efficiency, such as cell surface receptors, immune responses and so on. To establish stable cell lines, lentivirus is really an efficient way to for gene ORF expression, shRNA/miRNA and CRISPR/Cas9. However, despite mediating efficient transfection in both dividing and non-dividing cells, for some non-dividing primary cells, it is still difficult for lentivirus to transfect genes. Then, high-titer adenovirus to infect these cells like BMSC is recommended. While for in vivo experiment, the AAV viruses work best.

Genemedi got a rich experience in virus production, you could find more information and experience on https://www.genemedi.net/i/lentivirus-packaging; https://www.genemedi.net/i/adenovirus-packaging; https://www.genemedi.net/i/aav-packaging

Hope this may help you.

You can see some more cell transfection in different virus such as AAV, lentivirus and adenovirus: