Transfection is the process for experimentally delivering exogenous DNA, RNA, synthetic polynucleotides or oligonucleotides into cells in culture, without the uses of viral infection. MarinBio is expert in performing nucleic acid transfections using either biochemical, biological, or biophysical transfection methods and resulting in transient (expressed for limited times, e.g. days) or stable cell modifications (where the transgene is permanently integrated into the genome), enabling the desired new DNA-encoded or RNA-encoded gene functions and new protein expression in cultured cells.
In general, in the past 25 years one of the main purposes of transfection services for MarinBio clients has been to produce recombinant proteins or to specifically enhance or inhibit gene expression in transfected cells. Transfection is being used by many clients for the study of gene function, gene regulation, gene product (protein) regulation, for producing transgenic cells or organisms, and can be of high importance for client development of cell therapeutic or gene therapy products. Additionally, more and more clients are using gene therapy, whereby MarinBio develops a potency assay transfecting the client’s therapeutic DNA and measuring the newly expressed protein.
Transfection enables genomic, transciptomic, and proteomic studies of gene expression, gene modification (including gene editing), RNA transcription or RNA interference. At MarinBio our transfections of nucleic acids into cells are routinely performed with chemical transfection reagents to maintain excellent cell viability and peak cell performance in a broad range of cell types. MarinBio also adapts transfection protocols for multiple applications and can efficiently deliver plasmid DNA, siRNA/miRNA or CRISPR/Cas9 components. For clients needing a customized assay or for long term biologic protein drug release, MarinBio generates stable transfected cell clones and subclones, as well as cell banks. This includes hard to transfect cells, RNAi gene silencing, stem cell transfection, CRISPR/Cas genome editing, as well as in the manufacturing of antibodies, enzymes or other proteins.
Cells appear green when visualized by fluorescent microscopy after transfection of green fluorescent protein (GFP) and GFP protein expression.
The client’s time frame, product quantities and the final client goal is carefully considered by MarinBio in evaluating the requirements for transient or stable cell transfections.
Transiently transfected cells are typically harvested 2-4 days post transfection and are often used by clients for specific gene-encoded protein products, short-term gene RNA expression or for RNA interference (RNAi) in gene silencing, or for producing small scale recombinant protein manufacturing batches. Transient transfections with messenger RNA (mRNA) can have more rapid results (within minutes) due to mRNA expression in the cell cytoplasm, eliminating translocation to the nucleus and transcription. In many drug discovery applications, it is beneficial to quickly screen protein constructs with properly folded and/or glycosylated proteins using transient transfection methods, which allow simultaneous evaluation of various candidate molecules in less than one week.
Stable transfection yields long-term gene expression and requires screening and cloning cells for larger-scale protein production to minimize lot-to-lot variability used for longer-term pharmacology studies, gene therapy, or determining mechanisms of long- term gene regulation. In many instances, transient transfections are performed in parallel while more time intensive and resource intensive stable cell lines are under development and may require up to two to three months. At MarinBio stable transfected cell lines are used for clients requesting milligram-to-gram protein lots.
Transient transfection of large numbers of HEK293 or CHO suspension-adapted cells is available at MarinBio for rapid manufacture of relatively large amounts (e.g. milligrams per liter) of client protein in 3-7 days. MarinBio offers clients transient expression for rapid and ultra-high-yield protein production in mammalian cells based on high-density cell culture using cationic lipid-based transfections and has achieved high protein expression levels for many protein products, e.g. >1 g/L for Immunoglobulin G.
The professional scientists at MarinBio have significant expertise in a very wide variety of transfection methodologies. Over the past 25 years our expert scientists have made many hundreds of stable cell clones and lines expressing the client’s protein product. MarinBio has deep knowledge and experience with recombinant genes, transfection molecular biology, transgene expression, recombinant protein stability and modification (e.g. glycosylation), cell biology, and cell culture techniques. Thus, we successfully complete client projects on time and within budget, while providing weekly or more frequent updates. If you want to have your gene of interest tested, MarinBio successfully completes transfection studies, most often to provide the transfected cell clones selected to maximally express the desired protein product.