Landscape of in vitro cell transfection and in vivo transfection (siRNA transfection,DNA/plasmid transfection, nanoparticle transfecton, electroporation, transfection protocol and so on)
Introduction of transfection
In general, transfection is the process of delivering nucleic acids, such as DNA and RNA into eukaryotic cells, resulting in the expression or production of proteins or down-regulation of the targeted protein. Although not very common, protein transfection is also used to promote the rapid expression of target protein (such as Cas9 protein transfection for CRISPR for genome editing) [1,2]. To date, transfection has been applied in the field of basic research (gene overexpression or knockdown) and translational research (such as gene therapy [3], immunotherapy and induced pluripotent stem cell generation [4]).
Various delivery methods have been reported and each method has its specific properties. It’s very important to select the most effective transfection method according to the cell type, transfection efficiency, target cell viability and expression levels. Typically, the methods can be divided into two categories: non-viral and viral [5]. Non-viral transfection methods contain physical treatment (electroporation, nanoparticles, etc.), chemical materials mediated transfection (such as liposome, polymer). To deliver exogenous genes with viral methods, gene of interest should be packaged into a replication-deficient viral particle, and then the viral particles inject its DNA (including gene of interest) into target cells. To date, several types of virus have been applied for gene delivery, such as lentivirus, adenovirus (Ad), adeno-associated virus (AAV), retrovirus (Retro) and herpes simplex virus (HSV) and so on.
Table of Contents
Transfection in vitro
Transfection in vitro can be mediated by the non-viral reagents (such as polymer, liposome, nano-particle) or viral vectors (such as lentivirus, adenovirus and AAV).
Polymer-based transfection
Polymer transfection is a technique used to deliver DNA or RNA into cells with biodegradable cationic polymer [6], such as DEAE-dextran or polyethylenimine (PEI). The negatively charged DNA binds to the polycation and the complex is taken up by the cell via endocytosis. With some toxicity, polymer conjugated complexes can mediate transfection efficiently in some cell types, such as 293T cells, but are not suitable for transfection of sensitive cells and generation of stable cell lines.
Liposome-based transfection
Liposome transfection, also known as lipofection or lipid transfection, is a technology used to deliver DNA or RNA into cells mediated by liposomes, which are tiny vesicular structures with the same composition as the cell membrane and can easily fuse with the cell membrane [7]. To date, there have been several generations of liposome, differing in the addition of cationic lipid and DNA-compacting substances. Now the most commonly used liposome is highly potent cationic lipofection reagent that has been shown to effectively transfect plasmids or siRNA, as well as nucleic acid-protein complexes, into cultured adherent and suspension cell lines [8]. Besides, the efficiency of lipofection can be improved by treating transfected cells with a mild heat shock [9]. Genemedi has launched a transfection reagent of Lipogene, which is comparable to Lipofectamine 3000, much more effective than polyethylenimine (PEI).
You could find more information on this website: www.genemedi.com/i/lipogene-transfection-reagent.
Nanoparticle-mediated transfection
Non-viral gene delivery system – multifunctional envelope-type nano-devices (MEND) – has been proposed to package DNA or RNA into a nano-sized structure with programmed assembly of functional devices, enabling DNA protection from DNase, size control and improved packaging efficiency. Then polycations and lipids were added based on electrostatic interactions to realize lipid-film hydration [10]. Now Impalefection has been developed to impale cells to deliver DNA or RNA using nanomaterials, such as carbon nanofibers, nanotubes, nanorods, silicon nanowires [11]. Nano-particle transfection transfers the DNA or RNA via membrane fusion into the cell, avoiding DNA/RNA degradation by lysosome and showing great advantages over polymer and liposome.
Electroporation-mediated transfection
Electroporation, also known as gene electrotransfer, is a popular method to realize high efficient transfection. During electroporation, cells and DNA/RNA of interest are exposed to short pulses of an intense electric field, leading to a transient increase in cell membrane permeability and the entry of DNA/RNA around into cytoplasm. Once removing the electric field, the cell membrane stabilizes and closes, allowing the expression of the enclosed DNA/RNA. Though electroporation-mediated transfection is easily operated and reliable, but it leads to high rates of cell death and requires great numbers of cells. Besides, a special and matched electroporation device is needed for electroporation [12].
| Comparison | Polymer | Liposome | Aano-particle | Electroporation |
| Principle | Endocytosis | Endocytosis | Membrane fusion/impale cells | Transient increase in the permeability of cell membrane |
| Integration | No | No | No | No |
| Time to peak expression | 48h-72h | 48h-72h | 24h-48h | 48h-72h |
| Sustainable time | Transient expression | Transient expression | Transient expression | Transient expression |
| Cell Type | A number of cell types | Adherent and suspension cell lines | Almost all the cells | Not suitable for sensitive cell types |
| Particle diametre | 75 to 520 nm | 50-200nm | 100-300nm | – |
| Animal experiment | Low efficiency | Low efficiency | Target delivery | Target delivery |
| Cytotoxicity | High | Low | Non-toxic | – |
| Immune Response | No | No | No | No |
| Efficiency | Low efficiency (<10%) | Depend on cell type | High efficiency | High efficiency |
| Price | Inexpensive | Medium | Expensive | Most expensive (electroporation device) |
Lentivirus-based gene transduction in vitro
Lentiviral vectors can mediate efficient transfection and long-term expression of exogenous genes in both dividing and non-dividing cells and have been widely used for gene overexpression, RNA interference, microRNA research and in vivo animal experiments.
More useful information about lentivirus can be found on this website: https://www.genemedi.com/i/lentivirus-packaging .
Adenovirus-based gene transduction in vitro
Based on human adenovirus type 5 (Ad5), recombinant adenovirus (Ad) a replication-defective adenoviral vector system, is widely used for gene delivery in most cell types, and Genemedi got a rich experience in adenovirus packaging, you could find more information on https://www.genemedi.com/i/adenovirus-packaging .
AAV-based gene transduction in vitro
As the most excellent gene therapy vector, recombinant AAV can mediate long-term stable expression of target genes in vitro/vivo with broad range of host and low immunity. For instance, AAV-DJ/8 was engineered via DNA family shuffling technology, displaying better in vitro transduction efficiencies compared to that of other wild type serotypes and higher infectivity rates in various cells and tissue types [13]; and AAV6 shows great transduction efficiency in CD4+ and CD8+ T cells [14]. Genemedi is good at AAV production, you can find more information and protocols about AAV on this website: www.genemedi.com/i/aav-packaging .
Transfection in vivo
Transfection in vitro can be mediated by the non-viral reagents (such as liposome, polymer, nano-particle) or viral vectors (such as lentiviruses, adenovirus and AAV).
Non-viral methods
Formulations of liposome, polymer and nano-particle have been optimized for in vivo delivery, especially with the use of transfection enhancer and secondary injection. What’s more, the three non-viral methods can also be combined together (Figure 1). As shown in Figure 1, multifunctional envelope-type nano-devices (MEND) are designed to promote the delivery of DNA and RNA. In MEND, PEG is used to extend nucleic acids’ half-life in systemic circulation, ligands are for specific targeting, peptides, including a protein-transduction domain, are intend to increase intracellular availability, while membrane fusogenic lipids were utilized to enhance endosomal escape and protect the degradation of nucleic acids [10,15].
Over the last several years, many progresses have been made in the field of gene transfection with polymer, cationic liposome and nanoparticle. Parameters, such as charge density, and hydrophilic and hydrophobic content, are optimized to improve polymer characteristics (such as the strength of the polyplex–cell membrane interaction, serum stability, DNA release, endosomal escape and nuclear localization) [16]. Biocompatible, nontoxic and effective novel artificial liposomal systems are used in liposome-mediated gene transfection [17]. Magnetic nano-particles and implantable magnets are designed to deliver therapeutic gene to its target tissues, showing great advantages in clinic [18].
Viral vector
Lentiviruses can integrate a significant amount of viral cDNA into the host genome, mediate stable and long-term transgene expression, and efficiently infect dividing cells and nondividing cells in vivo [19]. You could find a lot of information and protocol about lentivirus on this website: https://www.genemedi.com/i/lentivirus-packaging.
With no integration ability, recombinant adenovirus can’t be integrated into host genome, have large cargo capacity (~8kb), and are easily manipulated with recombinant DNA techniques [20]. Genemedi got a rich experience in adenovirus packaging and purification, you could find more information on https://www.genemedi.com/i/aav-packaging.
With mild immunogenicity, adeno-associated viruses (AAV) have superior biosafety rating and broad range of infectivity and mediate long-term and stable expression of target gene in vivo [21]. Genemedi is good at lentivirus production, please find more information on this website: https://www.genemedi.com/i/aav-packaging.
| Comparison | Retrovirus | Lentivirus | Adenovirus | AAV |
| Genome | ss RNA | ss RNA | ds DNA | ss DNA |
| Integration | Yes | Yes | No | No |
| Packaging Capacity | 3kb | 4kb | 5.5kb | 2kb |
| Time to peak expression | 72h | 72h | 36h-72h | Cell: 7 days; Animals: 2 weeks |
| Sustainable time | About 3 weeks | Stable expression | Transient expression | > 6 months |
| Cell Type | Most Dividing | Most Dividing/Non-Dividing Cells | Most Dividing/Non-Dividing Cells | Most Dividing/Non-Dividing Cells |
| Titer | 10^7 TU/ml | 10^8 TU/ml | 10^11 PFU/ml | 10^12 vg/ml |
| Animal experiment | Suitable | Low efficiency | Lowest efficiency | Most suitable |
| Immune Response | High | Medium | Medium | Ultra-low |
Lentivirus, adenovirus and AAV all can mediate gene in vivo transfection. To date, lentiviral vector-based gene delivery in vivo has been proved to be effective in treatment of several diseases [22], including β-thalassemia [23], X-linked adrenoleukodystrophy (ALD) [24], metachromatic leukodystrophy [25,26], and Wiskott-Aldrich Syndrome [27]; adenovirus has been applied in treatment of several cancers, including prostate cancer [28], chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) [29], non-small cell lung cancer (NSCLC) [30], melanoma [31], renal cell carcinoma [32]; while AAV vectors have achieved promising gene therapy outcomes in a great number of diseases, including lipoprotein lipase deficiency (LPLD) [33], spinal muscular atrophy (SMA) [34], retinal dystrophy [35,36], cystic fibrosis [37,38], Duchenne Muscular Dystrophy [39], Hemophilia [40], congestive heart failure [41], Parkinson’s disease [42] and Rheumatoid Arthritis [43,44].
Small interfering RNA (siRNA) transfection
Small interfering RNA (siRNA) contains 21-25 nucleotides, specific for target RNA. Once entry into cells, siRNAs can be recruited into a multi-protein complex, known as RNA induced silencing complex (RISC), which interacts with the target RNA to mediate mRNA degradation, thus knock-down or suppress the expression of target gene [45]. siRNA transfection can be carried out with non-viral methods or viral vectors containing short hairpin RNA (shRNA), which can be subsequently processed into active siRNA.
siRNA transfection in vitro
siRNA transient transfection in vitro can be mediated by polymer and liposome. Stable transfection cell lines need to be mediated by viral vectors (lentivirus, adenovirus, AAV) carry shRNA targeting gene of interest. The knock-down of shRNA varies in different sequence and needs to be tested. Besides, shRNA should be cloned into viral vectors for virus production. Therefore, viral-based stable transfection takes so much time but presents little cytotoxicity and high efficiency.
siRNA transfection in vivo
Due to the instability of siRNA, low delivery efficiency and the superior difficulties in specific tissue distribution, in vivo siRNA transfection presents great technical challenges. Three methods have been adapted to deliver siRNA in vivo: 1) siRNAs with chemical modifications are generated, such as morpholino (MO) [47], 2′-deoxy-2′-fluoro-beta-D-arabinonucleic acid (FANA) [48], 2′-deoxy-2′-fluoro and 2′-O-methyl ribosugar modifications [49], Locked Nucleic Acid (LNA) modifications [50]; 2) encapsulation into liposomes also presents great protection from degradation in serum [51]; 3) viral vectors (lentivirus, adenovirus, AAV) containing shRNA targeting gene of interest show much higher delivery efficiency in vivo than chemical modifications or encapsulation, but these methods are time-consuming and might show some immunogenicity.
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