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Applications for Transfection

Transfection enables a wide variety of applications in addition to transient gene expression including:

It is important to consider both the efficiency of transfection and the level of cytotoxic effects when a transfection methodology or reagent is selected. Higher transfection efficiencies favor greater success whether the goal is to effectively knock down gene expression, create a stable cell line, increase virus titers, achieve maximum protein yields, or address stem cell research. However, a low-toxicity approach must not be overlooked, since highly cytotoxic approaches can lead to unwanted effects in the form of visible morphology changes, as well as unknown changes in gene expression or stress-response pathways.

Gene Silencing

The ability to silence genes plays an important role in molecular and cell biology and can be readily applied through transfection. Gene expression can be effectively reduced or eliminated by introducing small noncoding RNA molecules that inhibit RNA translation though a process termed RNA interference (RNAi). RNA molecules that take part in RNAi pathways include: (i) small interfering RNAs (siRNA), short (20–25 base pairs) double-stranded RNAs; and (ii) microRNAs (miRNAs), a separate class of short single-stranded RNAs (20–22 nucleotides). RNAi-based approaches rely on the inherent cellular machinery, shared among several eukaryotic organisms, to inhibit mRNA translation. RNAi pathways play in important role in regulating gene expression and are also believed to provide a mechanism to protect cells from extraneous nucleotide sequences (e.g., viruses and transposons).

RNAi pathways are elicited through cleavage of double-stranded RNAs (dsRNAs) to produce siRNAs, or by processing of noncoding RNAs to produce miRNAs. These separate RNAi pathways rely on cellular machinery such as the ribonuclease protein DICER and the RNA-induced silencing complex (RISC). DICER initiates the RNAi pathway by processing dsRNA to form siRNAs or mature miRNAs. These RNA molecules can bind to complementary sequences of mRNA within the RISC, and the mRNA can be cleaved by the catalytic component, Argonaute, which ultimately prevents translation.

Apart from this natural occurrence, exogenous sequences of siRNAs and miRNAs can be designed and introduced into cells through transfection to knock down relevant gene expression. This application serves as a tool to elucidate genetic pathways, determine protein function, or uncover new gene targets for biotherapeutic and pharmaceutical applications. Larger libraries of these RNA molecules are also available to perform larger genome RNAi analysis via high-throughput screening.

EXOGENOUS SEQUENCES OF siRNAs and miRNAs...

can be designed and introduced into cells through transfection to knock down relevant gene expression.

Because siRNA and miRNA differ in size and structure from plasmid DNA, transfection reagents can be optimized and formulated separately for delivery of these RNA molecules. In addition, delivery of siRNA and miRNA to the cytoplasm for incorporation into the RISC complex is sufficient for gene knockdown. Selection of the appropriate transfection methodology or reagent must first be considered, followed by further optimization for efficient siRNA and miRNA delivery and subsequent gene knockdown.

An alternative approach that takes advantage of RNAi to knock down gene expression is the use of short hairpin RNA (shRNA). These short RNA sequences can be expressed via viral or non-viral vectors. shRNA expression mimics a pathway similar to siRNA/miRNA since the expression product must be processed by DICER and ultimately incorporated into the RISC complex for targeted degradation of mRNA.

Although gene silencing via siRNA/miRNA and shRNA rely on similar RNAi pathways, the optimal method may depend on factors such as cell type, time constraints and transient vs. stable expression. A high and reliable level of knockdown can be achieved through siRNA transfection via a variety of quality transfection reagents and validated siRNA libraries. However, disadvantages with siRNA mediated knockdown are the chances of off-target effects and transient knockdown through siRNA dilution after multiple cell divisions. Alternatively, the use of shRNA can be carried out to generate stable knockdown cell lines, but this approach can be time-consuming, and cell types such as primary cells may yield lower transfection efficiencies through shRNA plasmid based transfection as opposed to siRNA delivery.

Another vehicle for shRNAs is viral transduction through adeno-associated virus (AAV), adenovirus and lentivirus. Expression through AAV or adenovirus can decrease the chance of insertional mutagenesis since these vectors are more likely to remain episomal, but this approach leads to more transient expression since the vectors are lost through multiple rounds of cell division. Lentivirus provides a stable solution through chromosomal integration, but this also presents the risk of insertional mutagenesis.

Regardless of whether shRNA is implemented via viral or non-viral methods, transfection plays a role to facilitate gene silencing via this approach. Non-viral vectors can be introduced via chemical reagents optimized for plasmid transfection, or they can be delivered via electroporation. For viral based approaches, virus generation can be carried out via transfection (see Virus Production). Under either delivery, shRNA mediated knockdown can provide a more stable means of RNAi than siRNA/miRNA with less turnover.

Whether shRNA, siRNA or miRNA mediated RNAi approaches are implemented, the overall goal of the experiment needs to be established. One must also consider the gene targets and cell types used, design the proper sequence for specificity, determine duration of expression and select the most effective means of delivery to ensure success.

For more information on transfection for RNAi applications, additional resources can be found on the Mirus Bio website:

Optimize siRNA Transfection

Deliver microRNA (miRNA) Effectively

Reverse Transfection Protocol for siRNA/miRNA

siRNA Mediated Pathway

siRNA Mediated Pathway

miRNA Mediated Pathway

miRNA Mediated Pathway

shRNA Mediated Pathway

shRNA Mediated Pathway

Generation of Stable Cell Lines

Generation of Stable Cell Lines

The creation of a stable cell line provides many advantages over transient gene expression, such as the elimination of the need for repeated transfections, establishment of more uniform gene expression within a cell population, and provision of a system for long-term experiments. These experiments may include prolonged expression of a target gene or permanent silencing of a particular gene.

To generate a stable cell line, introduced DNA must integrate into the host genome, which can be a rare event. This process requires patience and diligence in addition to quality transfection systems. Creating a stable cell line requires the following:

  • Delivery to the cell
  • Inclusion into the nucleus
  • Integration into the chromosome

Integration into the chromosome is typically accomplished through the inclusion of a homologous DNA sequence that flanks the inserted DNA element.

STABLE CELL LINE GENERATION...

eliminates the need for repeated transfections, establishes a more uniform gene expression within a cell population, and provision of a system for long-term experiments.

Very high transfection efficiency is required in order to maximize the chance of a rare chromosomal integration event. Additionally, a positive selection marker such as an antibiotic (e.g., hygromycin, neomycin, or zeocin) is required for proper selection of a stable cell line expressing the gene of interest. The use of a linearized plasmid can also increase the likelihood of integration of the gene of interest.

Following multiple rounds of selection to isolate cells with the integrated plasmid containing the drug resistance gene and the gene of interest, a mixed population of drug-resistant cells can be used directly for further clonal isolation. The use of a mixed population, or batch culture, saves time but suffers from the disadvantage of using an undefined and genotypically mixed culture. A clonal isolate can be generated using serial dilution plating to isolate single cells or by picking individual colonies with rings or pipettes. The selection process is then applied to these clonal isolates to verify integration.

Final verification of the stable cell line typically includes an assay to verify expression of the gene of interest. These methods include detection through Western blot and sequencing of genomic DNA to verify integration.

More details on stable cell line generation can be found at our Tips from the Bench: Transfection Tip – Generate Stable Cell Lines.

Virus Production

Virus Production

Virus applications from basic virology research to viral transduction and gene therapies all require the ability to develop high titers of functional virus. Recombinant viruses can be generated to study all aspects of virology including viral life cycles, structure, and mode of infection. Viruses provide an alternative to nucleic acid delivery when a specific cell type is refractory to chemical transfection or electroporation methods. Gene therapies have also called upon viral vectors to introduce genes into cells in vivo . Regardless of the application, transfection is a helpful tool that is commonly used to create functional viruses.

VIRUSES PROVIDE...

an alternative to nucleic acid delivery when a specific cell type is refractory to chemical transfection or electroporation methods… regardless of the application, transfection is a helpful tool that is commonly used to create functional viruses.

Whether the virus is needed for viral transduction purposes or for the study of the virus itself — its life cycle,structure, or health risk, an essential requirement is the generation of a functional virus in sufficient amounts. This process entails delivery of genetic material in the form of DNA or RNA that contains the blueprint for the viral life cycle, including replication, packaging, and — in some cases — genomic integration. Viral transduction is still a commonly used methodology to deliver genetic material into cells when the following apply:

  • Viral transduction is not a safety or regulatory concern.
  • Cells are refractory to other nucleic acid delivery methods (e.g., chemical transfection or electroporation).
  • Higher transfection efficiencies are necessary compared to those afforded by chemical transfection.

Commonly Used Viruses and Applications

Virus Nucleic Acid Characteristics Examples
Retrovirus RNA - Integrative (random)
- Replication-competent or replication-defective
- Requires actively dividing cells for infection
MMLV – Moloney murine leukemia virus
Lentivirus RNA - Integrative (random)
- Can infect nondividing cells
- HIV – Human immunodeficiency virus
- SIV – Simian immunodeficiency virus
- FIV – Feline immunodeficiency virus
Adenovirus DNA - Non-integrative
- Requires actively dividing cells for infection
Various; depend on host and type

- HAdV-B and C (Human respiratory disease)
- HAdV-B and D (Human conjunctivitis)
- HAdV-F types 40 and 41 (Human gastroenteritis)
- CadV-2 (Canine adenovirus)
- Equine adenovirus 1 (Horse adenovirus)
Adeno-associated
viruses (AAV)
DNA - Integrative (wildtype is site-specific)
- Can infect nondividing cells
- Nonpathogenic
AAVS1 (wildtype integrates at chromosome 19)

Transient transfection is part of the general workflow for the production of most viruses, but the details will be specific to each type of virus produced. Stable cell lines can also be generated to actively produce viral vectors that are not included in the Table 1. The following key steps are characteristic of virus generation through transient transfection:

Nucleic Acid Preparation » Transfection » Viral Harvest » Titer

1. Nucleic Acid Preparation

In most cases, it is necessary to co-transfect two or more plasmids—the vector plasmid and one or more helper plasmids. The vector plasmid contains the gene of interest designed for homologous recombination; the helper (i.e., packaging) plasmid(s) encodes the necessary structural, regulatory, and replication genes to produce functional virus. Together, these plasmids will generate a virus that can transduce the gene of interest for subsequent genomic integration.

2. Transfection

Multiple plasmids can be transiently transfected through the use of calcium phosphate or chemical transfection reagents. Calcium phosphate provides an economical means for generating virus in this manner but can also exhibit low reproducibility and low virus titer yields. Chemical transfection reagents are employed for more consistency and higher virus titers.

A packaging cell line is also required for generating the viral vector. In many cases, the human embryonic kidney cell line, HEK 293, or derivatives such as HEK 293T or HEK 293A, can be suitable hosts since they readily expresses a variety of viral genes. HEK 293 cells can be transfected readily, and—in the cases of the derivative cell lines—may be deficient in packaging virus without the use of a helper plasmid (e.g.,HEK 293T for retrovirus) or may produce a necessary viral protein (e.g., HEK 293A for adenovirus).

When using chemical transfection reagents for DNA delivery, the total amount of DNA required from all plasmid sources must be considered in order to determine the optimal amount of reagent for high-efficiency transfection.

For example, if a 3:1 ratio of µl of transfection reagent per µg of DNA is optimal for high-efficiency transfection in the packaging cell line, and 3 µg of vector plasmid and 1 µg of helper plasmids are necessary (i.e., 4 µg of DNA total), then a total of 12 µl of reagent will be needed for efficient transfection. Complex formation is facilitated by incubation of the reagent and DNA for 15 to 30 minutes. Complexes are then added directly to cells for transfection

3. Harvest

After transfection, harvest of the virus can vary depending on the virus produced and the protocol followed. Protocols can require multiple harvests of supernatant and medium changes within 24 to 48 hours after transfection. Harvests are typically filtered, aliquoted, and stored at –80°C.

4. Titer

It is generally recommended to titer the virus in order to determine the effective concentration for infection. Viral titers are determined by infecting cell stocks with serial dilutions of the stock virus. After infection, most viral vectors contain a fluorescent reporter to quantify infection through flow cytometry analysis.

Viral transduction provides a powerful and effective means to deliver nucleic acids into a variety of cell types, including primary cells. However, in order to produce virus, introduction of viral genes into a packaging host is still necessary. Transfection provides a robust and reproducible means to generate high-titer yields in contrast to calcium phosphate.

Virus Production Process

1. Nucleic Acid Preparation

2. Transfection

3. Harvest

4. Titer

For more information on the use of transfection for high-titer virus production, additional resources can be found on the Mirus Bio website:

TransIT® Reagents for High-titer Virus Production

High-Titer Production of Recombinant Lentivirus in HEK 293FT Cells: Calcium phosphate vs. Lipopolyplex Transfection VIEW PDF

Large-scale Protein Production

Wave Bag

Transient transfection can be used in mammalian cell-culture systems to generate high yields of functional protein for biotherapeutic applications. Mammalian cell lines, such as Chinese hamster ovary (CHO) and human embryonic kidney (HEK 293) cells, are attractive hosts for biotherapeutic protein production because they provide the correct post-translational modifications for biologically relevant use. In addition, these cell lines are well characterized and are already amenable to Food and Drug Administration (FDA) clearance.

During the cumbersome process of generating a suitable stable cell line for biotherapeutic protein production, which can take several weeks or even months, large amounts of protein can be produced from suspension CHO or HEK 293 cells via transient transfection. Effective use of transient transfection for large-scale protein production requires a reagent that can transfect in multiple media formulations, can be effectively scaled up to transfect larger volumes, and can reproducibly generate large amounts of protein. In addition, the formulation of such a reagent should be animal product-free to address any Food and Drug Administration (FDA) regulatory concerns.

EFFECTIVE USE OF TRANSIENT TRANSFECTION...

for large-scale protein production requires a reagent that can transfect in multiple media formulations, can be effectively scaled up to transfect larger volumes, and can reproducibly generate large amounts of protein.

One key consideration in such large-scale applications can also be cost. Linear PEI is a seemingly cost-effective option for carrying out large-scale transfections in suspension CHO and HEK 293 cells. However, this compound is incompatible with multiple media formulations and is not as effective at generating high protein yields as other transfection reagents. Low protein yields will increase the cost of protein production because of the time, wages, and materials associated with producing comparable protein amounts.

For more information on large-scale protein production in suspension cells, additional resources can be found on the Mirus Bio website:

Selecting a Transfection Reagent for Large Scale Protein Production in Suspension 293 Cell Types VIEW PDF

Maximize Protein Expression in CHO Suspension Cells VIEW PDF

Stem Cell Applications

Stem cells hold the promise of revolutionizing therapy for a myriad of diseases. Biologically relevant models can be generated through reprogramming and differentiation. These techniques can be accomplished via the introduction of transcription factors through the use of small molecules, recombinant virus transduction, or transfection of proteins or nucleic acid. Transfection provides a nonviral approach that is more efficient than protein transfection and safer than the use of viruses for infection.

Entry Points for Transfection

Entry Points for Transfection

Entry Points for Transfection

Transfection can be used at many points throughout the course of a reprogramming or stem cell differentiation experiment. Transfection of nucleic acids, such as plasmid DNA, mRNA, and siRNA or miRNA, serve as vital tools in stem cell reprogramming and differentiation. Adult fibroblast cells can be transfected or transduced via several methods (e.g., recombinant virus, plasmid, protein, mRNA, small molecules, or miRNA) with a combination of transcription factors, including KLF4, SOX2, c-MYC, NANOG, OCT-4, or LIN-28, to reprogram the cells to a pluripotent state. Induced pluripotent stem (iPS) cells can then be differentiated to a myriad of cell types through growth factor addition and/or transfection of selection markers driven by cell type-specific promoters. Cell types derived from stem cells—such as cardiomyocytes, adipocytes, neural cells, pancreatic beta cells, and hematopoietic progenitor cells—provide researchers with relevant models for their experiments.

For more information and examples highlighting transfection for stem cell applications, additional resources can be found on the Mirus Bio website:

From Reprogramming to Differentiation – Transfection Applications for Stem Cell Research VIEW PDF

High Efficiency Transfection of iCell® Cardiomyocytes (Cellular Dynamics International, Inc.) and Stem cell Relevant Sources VIEW PDF

Optimized transfection protocol for iCell® Cardiomyocytes (Cellular Dynamics International, Inc.) using TransIT-TKO® Transfection Reagent: VIEW PROTOCOL PDF