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Transfection Reagents - Powerful Tools to Enable Genetic Manipulation

Apr 02, 2015 at 12:00 PM ET

Abstract

Transfection technology allows for transient genetic manipulation in cell cultures that is quick, reliable, and minimally toxic. Lipid-based transfection reagents are powerful tools to investigate the role of various genes in cellular physiology. Gene expression may be enhanced by delivering DNA or mRNA for specific genes or gene products into cells. In addition, gene knockdown may be achieved through transfection of small RNA constructs, which utilize the RNAi pathway to inhibit gene expression. This webinar will provide an introduction to transfection, an overview of current transfection approaches, and a brief guide to best practices in the design and optimization of transfection experiments. Critical aspects include the cell type, culture conditions, and design of nucleic constructs. We will then focus on ATCC’s highly efficient transfection reagents, which have been optimized in a broad spectrum of cell types for a range of applications, including gene expression and siRNA-mediated knockdown.

Presenter

James Clinton, headshot.

James Clinton, PhD

Senior Scientist, ATCC

James Clinton, Ph.D., works in new product development, with a focus on primary cells and advanced, physiologically relevant culture systems using novel technologies. Previously he worked at University of California, San Diego and the La Jolla Institute for Molecular Medicine. Dr. Clinton attended Washington State University and University of California, San Diego where he studied Neuroscience.

Questions and Answers

Are HeLa cells (ATCC CCL-2) harder to transfect than HEK293-T17 cells (ATCC CRL11268)?

In general, we find both cell lines to be equally amenable to transfection, and both cell lines exhibited >90% transfection efficiency with the ATCC TransfeX reagent (ATCC ACS-4005).

Are samples of the transfection reagents available?

Yes, free samples of ATCC transfection reagents are available for the cost of shipping. Please visit www.atcc.org/transfection to request a free sample*.

Can siFEX be stored at -20°C?

We recommend storing the reagent siFEX (ATCC ACS-4006) at 4°C. Our testing suggests that a single freeze/thaw will not immediately impact performance of the reagent; however, we do not know whether storage at this temperature will affect its performance long-term.

Can the GeneXPlus Transfection Reagent (ATCC ACS-4004) be used on LCL 8664 rhesus monkey lymphoma cells (ATCC CRL-1805)?

The GeneXPlus Transfection Reagent (ATCC ACS-4004) works well with other suspension cell lines and may be tried with the LCL 8664 cells; however, ATCC has not tested the GeneXPlus reagent specifically on these cells.

Do you provide cell-line specific reverse transfection protocols?

ATCC provides a standardized forward transfection protocol. In reverse transfections, cells are directly seeded into wells already containing the transfection complex, therefore combining two steps in the protocol workflow and effectively saving 1 day. However, in some cases, reverse transfection may increase cytotoxicity and thus necessitate seeding a higher number of cells initially. This effect will vary with the type of cells being transfected.

Have you used any of the transfection reagents on JEG-3 cells (ATCC HTB-36)?

ATCC has not tested any of our transfection reagents with JEG-3 cells. Please visit the ATCC website at www.atcc.org/transfection to request a sample of an ATCC transfection reagent if you would like to perform your own testing.

Is a post-transfection media change required?

We include a 24 h media change post-transfection as an optional step on most of our transfection protocols. In many cases it is not necessary, but it can significantly reduce Pa cytotoxicity in some cell types. We have also found that a media change at 6 h or 8 h is sufficient to achieve the same effect (for example, transfecting cells in the morning and performing the media change in the late afternoon).

What is the benefit of using linearized plasmid for stable transfections?

Using linearized DNA avoids the possibility that your selection marker will be cut during integration into the host cell genome, thereby resulting in a greater efficiency of positive clone generation.

What is the maximum confluence at which cells may be optimally transfected?

If the cells you are working with are not contact inhibited and continue to proliferate when confluent, in theory, there is no maximum. However, it is likely that post-confluent cultures will not transfect as well as sub-confluent cultures, simply due to penetration and insufficient exposure of the cells to the transfection complexes. We recommend that the cells should be in the log phase of their growth curve, regardless of confluence, to ensure high transfection efficiencies.

What mRNA did you use for transfection with TransfeX (ATCC ACS-4005)?

For mRNA transfection with the ATCC TransfeX reagent, we used an EGFP expressing mRNA (TriLink Biotechnologies), which is capped, polyadenylated, and chemically modified with 5- methylcytidine and pseudouridine.

What reagent would you recommend for transfecting HL-60 cells (ATCC CCL-240)?

HL-60 is a suspension cell line derived from blood. These cell types are typically very difficult to transfect using lipid-based reagents. None of the reagents we have tested (ATCC as well as alternative suppliers) produced efficiencies of greater than 5%. We therefore suggest using electroporation or other non-lipid based methods to transfect HL-60 cells.

What should ATCC transfection reagents be diluted with?

We only recommend diluting ATCC reagents if the volume necessary is too small to pipette accurately. In that case, we suggest diluting the reagent in serum-free media immediately before transfection and discarding any that is unused. We do not recommend storing diluted reagent for later use.

Why are validated control nucleotides important in transfection?

Validated control nucleic acid sequences, whether it is a plasmid that expresses a fluorescent protein or a siRNA that effectively knocks down a house-keeping gene such as GAPDH, are vital for optimizing and troubleshooting experiments involving transfection.