Specially designed cationic lipids, such as the Lipofectamine® Transfection Reagents, facilitate DNA and siRNA delivery into cells (Chesnoy and Huang, 2000; Hirko et al., 2003; Liu et al., 2003). The basic structure of cationic lipids consists of a positively charged head group and one or two hydrocarbon chains. The charged head group governs the interaction between the lipid and the phosphate backbone of the nucleic acid, and facilitates DNA condensation. Often, cationic lipids are formulated with a neutral co-lipid or helper lipid, followed by extrusion or microfluidization, which results in a unilamellar liposomal structure with a positive surface charge when in water.

The positive surface charge of the liposomes mediates the interaction of the nucleic acid and the cell membrane, allowing for fusion of the liposome/nucleic acid transfection complex with the negatively charged cell membrane. The transfection complex is thought to enter the cell through endocytosis.

Endocytosis is the process where a localized region of the cellular membrane uptakes the DNA:liposome complex by forming a membrane bound/intracellular vesicle.

Once inside the cell, the complex must escape the endosomal pathway, diffuse through the cytoplasm, and enter the nucleus for gene expression. Cationic lipids are thought to facilitate transfection during the early steps of the process by mediating DNA condensation and DNA/cellular interactions.

Figure 1: Mechanism of cationic lipid-mediated delivery

Problems with traditional methods

Methods like calcium phosphate co-precipitation, DEAE-dextran, polybrene, and electroporation include problems such as:

  • low efficiency of DNA delivery
  • poor reproducibility
  • cell toxicity
  • inconvenience

In contrast, lipid mediated transfection:

  • Yields high and previously unattainable transfection efficiencies
  • Works in a wide variety of eukaryotic cells
  • Is simple to perform
  • Ensures consistently reproducible results

Moreover, a number of cell lines normally resistant to transfection by other methods transfect successfully with cationic lipid reagents.

The principle of delivery using cationic lipid reagents thus differs from prior attempts to use neutral liposomes for transfections. With cationic lipid reagents, the DNA solution is not deliberately encapsulated within the liposomes; rather, the negatively charged DNA binds spontaneously to the positively charged liposomes, forming DNA cationic lipid reagent complexes.

Cationic lipid transfection reagents

Cationic lipid-mediated delivery is a fast, simple, and reproducible means for easily introducing DNA, RNA, siRNA, or oligonucleotides into eukaryotic cells. It allows the highly efficient transfection of a broad range of cell types, including adherent, suspension, and insect cells, as well as primary cultures. When selecting a transfection reagent, you must consider the payload you wish to deliver (DNA, RNA, or protein) and the type of cellsyou want to transfect, because the choice of the transfection reagent strongly influences transfections results.

The tables below lists the key features and applications of various cationic-lipid transfection reagents available from Life Technologies™. Click here for more information on each transfection reagent and for optimized transfection protocols for a wide range of cell lines.

Trasfection reagent Payload Key features & applications
Lipofectamine® 3000
  • Highest efficiency and expression results for plasmid and RNAi cotransfections
  • High transfection efficiency even at low doses
  • Works effectively with a wide range of cell types, both adherent and suspension
  • No need for wash steps before or after transfection
  • Complexes can be added to cells growing in serum-containing media
Lipofectamine® 2000
  • High efficiency and expression results for plasmid or RNAi transfections
  • Works effectively with a wide range of cell types, both adherent and suspension
  • For robust cells
  • No need for wash steps before or after transfection
  • Complexes can be added to cells growing in serum-containing media
  • Ideal at >90% confluency at the time of transfection
  • Recommended for delivery of Stealth RNAi® and Silencer® Select siRNAs, dicer-generated siRNA pools and plasmids containing shRNA cassettes
  • High-throughput
Lipofectamine® 2000 CD
  • Same performance as Lipofectamine® 2000, certified animal-origin free (“CD” = chemically defined)
Lipofectamine® LTX with PLUS™ Reagent
  • Highest transfection efficiency in Chinese Hamster Ovary (CHO) cells
  • High transfection efficiency and significantly lower toxicity for a wide range of cell lines
  • Very gentle to cells
  • Wide range of cell lines, including primary and disease-related cells
  • High protein expression
  • Delivery of shRNA and miR RNAi vectors
  • Significantly improved transfection performance in a number of primary and hard-to-transfect cell lines
  • Optimized protocols for over 30 different cell lines are available, so much less time is needed for evaluation and optimization
Lipofectamine®
  • First generation reagent for plasmid DNA transfections
Lipofectin®
  • First generation reagent for plasmid DNA transfections
Trasfection reagent Payload Key features & applications
Lipofectamine® RNAiMAX
  • Recommended for transient delivery of Stealth RNAi® and Silencer® Select siRNAs, dicer-generated siRNA pools, mirVana™ miRNA Mimics and Inhibitors, mRNA, and snRNA
  • Requires lower RNAi concentrations leading to more effective gene knockdown with minimal non-specific effects
  • Minimal cytotoxicity across a 10-fold concentration range of transfection reagent
  • Compatibility with a broad range of cell types
  • High-throughput
  • Highest knockdown with less RNAi
  • in vitro transfection
Lipofectamine® MessengerMAX
  • Highest transfection efficiency in neurons (> 2-fold) and a broad spectrum of primary cell types
  • Enhanced gene editing outcomes using mRNA CRISPRs
  • Faster protein expression with no risk of genomic integration
Invivofectamine® 2.0
  • Efficiently delivers siRNA in vivothrough systemic delivery
  • Highly effective mRNA, protein, and functional knockdown
  • Low toxicity profile and easy to use
Oligofectamine®
  • Transfection of antisense oligonucleotides
  • High-throughput
  • Highly-specific, non-toxic
  • Ideal for low confluency (30–50% confluent at the time of transfection)
Trasfection reagent Payload Key features & applications
ExpiFectamine® 293
  • Transfection of high density 293 suspension cell culture for bioproduction
  • Transfection enhancers boost performance and protein expression with yields 2- to 10-fold higher than other transfection reagents used on high density 293 cell cultures
  • Robust and reproducible transfection results
  • Scale transfections for culture volumes of less than 1 mL to greater than 10 liters, while maintaining equivalent volumetric protein yields
FreeStyle™ MAX
  • Used for rapid large-scale transient mammalian protein expression for bioproduction
  • High-yield production with milligrams of protein yield
  • Optimized for transient transfection in CHO suspension cells and also works for HEK-293 cells
293fectin™
  • Used for transient protein bioproduction in combination with the FreeStyle™ 293 Expression System
  • Optimized for suspension FreeStyle™ 293-F cells
Optifect™
  • Ideal for low confluency (<70% confluent at the time of transfection)
  • For cell lines that are sensitive to transfection reagents
Cellfectin®
  • Optimal transfection of insect cells, including S2, Sf9, Sf21 and High Five™ cells
DMRIE-C Reagent
  • Transfection of suspension cells, including CHO, lymphoid and Jurkat cell lines
Symbol Explanation Symbol Explanation
Plasmid DNA for expression of
protein, shRNA, and miRNA
Plasmid DNA for expression of
protein
Non-coding RNA for RNAi
inhibition of gene expression
mRNA for expression of protein
Co-delivery for cotransfection of
RNAi vectors and siRNAs
Oligonucleotides for antisense
inhibition of gene expression