Can you transfect primary cells?

Transfection of primary cells and stem cells is a problem in the laboratory routine and further in tissue engineering and gene therapy. Most methods working effectively for cell lines in culture fail to transfect primary cells.

How do you transfect cell protocols?

In a standard transfection protocol, the cells are plated on day 1, transfected on day 2 and assayed on day 3 or 4. In a reverse transfection protocol, cells are added directly to a plate containing the transfection reagent/DNA mix and assayed on day 2 or 3.

Does the type of cell line used matter in transfection?

However, if the purpose of the transfection experiment is high-level production of recombinant proteins, it is not important that the cell line represents the in vivo situation as long as the cell line can express sufficient quantities of recombinant proteins with proper folding and post-translational modifications.

Why is it difficult to transfect primary cells?

Several types of cells (primary cells, stem cells or cancer cell lines) remain difficult to transfect with plasmid DNA. This is mainly due to their fragility and slow-dividing rates.

What is the best transfection method?

Magnetofection method
Up to now, the best method for transfecting primary cells (and hard-to-transfect cells) is the Magnetofection method. Briefly, Magnetofection uses a magnetic field to concentrate magnetic complexes onto the cell surface.

How do transfection reagents work?

Transfection reagents, such as Transfectamine™ 5000, are positively charged and attract the negatively charged DNA to form a positively charged polymer, which can interact with negatively charged cell membrane which enables the uptake of this polymer into the cell.

What is PEI used for in transfection?

The polycation polyethylenimine (PEI) is one of the most utilized reagents for small- to large-scale transfections as it is simple to use and, when combined with optimized expression vectors and cell lines, provides high transfection efficiency and titers.

Does transfection change DNA?

While DNA-based vectors (viruses, plasmids) that encode a short RNA molecule can also be used, short-RNA transfection does not risk modification of the cell’s DNA, a characteristic that has led to the development of short RNA as a new class of macromolecular drugs.

What is transfection virus?

Viral Transfection (Viral Transduction) This method involves the use of viral vectors to deliver nucleic acids into cells. Viral delivery systems such as lentiviral, adenoviral and oncoretroviral vectors can be used for transferring nucleic acids, even in hard-to-transfect cells.

Can you transfect confluent cells?

Do not allow the cells to remain confluent for more than 24 hours. The optimal cell density for transfection varies for different cell types, applications, and transfection technology, and should be determined for every new cell line to be transfected.

What is the best protocol for cell-specific transfection?

Cell-Specific Transfection Protocols NOTE: Invitrogen Lipofectamine Transfection Reagent Protocols have been optimized for efficiency, viability, and reproducibility across a broad range of cell types. This is often the best place to start, especially in a new cell line.

Is viral transduction the best way to transfect primary cells?

As compared to non-viral transfection, viral transduction is widely recognized as a highly effective method to transfect difficult-to-transfect cells such as primary cells ( Mali, 2013; Wang, Shang & Li, 2015 ).

What is transfection and how does it work?

Transfection is a process by which foreign nucleic acids are delivered into a eukaryotic cell to modify the host cell’s genetic makeup ( Kim & Eberwine, 2010; Chow et al., 2016 ).

Which cell lines can be transfected with exogenous nucleic acids?

Many cultured cell lines, as well as the majority of primary cell cultures, are able to be transfected with exogenous nucleic acids when appropriate transfection approaches are employed.