Transfection
See also: RNA transfection and RNA vaccines
Transfection is the process of deliberately introducing naked or purified nucleic acids into eukaryotic cells.[1][2] It may also refer to other methods and cell types, although other terms are often preferred: "transformation" is typically used to describe non-viral DNA transfer in bacteria and non-animal eukaryotic cells, including plant cells. In animal cells, transfection is the preferred term as transformation is also used to refer to progression to a cancerous state (carcinogenesis) in these cells. Transduction is often used to describe virus-mediated gene transfer into eukaryotic cells.[2][3]
The word transfection is a portmanteau of trans- and infection. Genetic material (such as supercoiled plasmid DNA or siRNA constructs), or even proteins such as antibodies, may be transfected. Transfection of animal cells typically involves opening transient pores or "holes" in the cell membrane to allow the uptake of material. Transfection can be carried out using calcium phosphate (i.e. tricalcium phosphate), by electroporation, by cell squeezing, or by mixing a cationic lipid with the material to produce liposomes that fuse with the cell membrane and deposit their cargo inside.
Transfection can result in unexpected morphologies and abnormalities in target cells.
Contents
The meaning of the term has evolved.[4] The original meaning of transfection was "infection by transformation", i.e., introduction of genetic material, DNA or RNA, from a prokaryote-infecting virus or bacteriophage into cells, resulting in an infection. Because the term transformation had another sense in animal cell biology (a genetic change allowing long-term propagation in culture, or acquisition of properties typical of cancer cells), the term transfection acquired, for animal cells, its present meaning of a change in cell properties caused by introduction of DNA.
There are various methods of introducing foreign DNA into a eukaryotic cell: some rely on physical treatment (electroporation, cell squeezing, nanoparticles, magnetofection); others rely on chemical materials or biological particles (viruses) that are used as carriers. Gene delivery is, for example, one of the steps necessary for gene therapy and the genetic modification of crops. There are many different methods of gene delivery developed for various types of cells and tissues, from bacterial to mammalian. Generally, the methods can be divided into two categories: non-viral and viral.[5]
Non-viral methods include physical methods such as electroporation, microinjection, gene gun, impalefection, hydrostatic pressure, continuous infusion, and sonication and chemical, such as lipofection, which is a lipid-mediated DNA-transfection process utilizing liposome vectors. It can also include the use of polymeric gene carriers (polyplexes).[6]
Virus mediated gene delivery utilizes the ability of a virus to inject its DNA inside a host cell. A gene that is intended for delivery is packaged into a replication-deficient viral particle. Viruses used to date include retrovirus, lentivirus, adenovirus, adeno-associated virus, and herpes simplex virus. However, there are drawbacks to using viruses to deliver genes into cells. Viruses can only deliver very small pieces of DNA into the cells, it is labor-intensive and there are risks of random insertion sites, cytopathic effects and mutagenesis.
Bacterial spheroplasts can transfect animal cells.
Chemical-based transfection[edit]
Chemical-based transfection can be divided into several kinds: cyclodextrin,[7] polymers,[8] liposomes, or nanoparticles[9] (with or without chemical or viral functionalization. See below).
One of the cheapest methods uses calcium phosphate, originally discovered by F. L. Graham and A. J. van der Eb in 1973[10] (see also[11]). HEPES-buffered saline solution (HeBS) containing phosphate ions is combined with a calcium chloride solution containing the DNA to be transfected. When the two are combined, a fine precipitate of the positively charged calcium and the negatively charged phosphate will form, binding the DNA to be transfected on its surface. The suspension of the precipitate is then added to the cells to be transfected (usually a cell culture grown in a monolayer). By a process not entirely understood, the cells take up some of the precipitate, and with it, the DNA. This process has been a preferred method of identifying many oncogenes.[12]
Another method is the use of cationic polymers 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.
Lipofection (or liposome transfection) is a technique used to inject genetic material into a cell by means of liposomes, which are vesicles that can easily merge with the cell membrane since they are both made of a phospholipid bilayer.[13] Lipofection generally uses a positively charged (cationic) lipid (cationic liposomes or mixtures) to form an aggregate with the negatively charged (anionic) genetic material.[14] This transfection technology performs the same tasks as other biochemical procedures utilizing polymers, DEAE-dextran, calcium phosphate, and electroporation. The efficiency of lipofection can be improved by treating transfected cells with a mild heat shock.[15]
Fugene is a series of widely used proprietary non-liposomal transfection reagents capable of directly transfecting a wide variety of cells with high efficiency and low toxicity.[16][17][18][19]
Dendrimer is a class of highly branched molecules based on various building blocks and synthesized through a convergent or a divergent method. These dendrimers bind the nucleic acids to form dendriplexes that then penetrate the cells.[20][21]
Non-chemical methods[edit]
Electroporator with square wave and exponential decay waveforms for in vitro, in vivo, adherent cell and 96 well electroporation applications. Manufactured by BTX Harvard Apparatus, Holliston MA USA.
Electroporation (gene electrotransfer) is a popular method, where transient increase in the permeability of cell membrane is achieved when the cells are exposed to short pulses of an intense electric field.
Cell squeezing is a method invented in 2012 by Armon Sharei, Robert Langer and Klavs Jensen at MIT. It enables delivery of molecules into cells via cell membrane deformation. It is a high throughput vector-free microfluidic platform for intracellular delivery. It reduces the possibility of toxicity or off-target effects as it does not rely on exogenous materials or electrical fields.[22]
Sonoporation uses high-intensity ultrasound to induce pore formation in cell membranes. This pore formation is attributed mainly to the cavitation of gas bubbles interacting with nearby cell membranes since it is enhanced by the addition of ultrasound contrast agent, a source of cavitation nuclei.
Optical transfection is a method where a tiny (~1 µm diameter) hole is transiently generated in the plasma membrane of a cell using a highly focused laser. This technique was first described in 1984 by Tsukakoshi et al., who used a frequency tripled Nd:YAG to generate stable and transient transfection of normal rat kidney cells.[23] In this technique, one cell at a time is treated, making it particularly useful for single cell analysis.
Protoplast fusion is a technique in which transformed bacterial cells are treated with lysozyme in order to remove the cell wall. Following this, fusogenic agents (e.g., Sendai virus, PEG, electroporation) are used in order to fuse the protoplast carrying the gene of interest with the target recipient cell. A major disadvantage of this method is that bacterial components are non-specifically introduced into the target cell as well.
Impalefection is a method of introducing DNA bound to a surface of a nanofiber that is inserted into a cell. This approach can also be implemented with arrays of nanofibers that are introduced into large numbers of cells and intact tissue.
Hydrodynamic delivery is a method used in mice and rats, but to a lesser extent in larger animals, in which DNA most often in plasmids (including transposons) can be delivered to the liver using hydrodynamic injection that involves infusion of a relatively large volume in the blood in less than 10 seconds; nearly all of the DNA is expressed in the liver by this procedure.[24][25][26]
Particle-based methods[edit]
A direct approach to transfection is the gene gun, where the DNA is coupled to a nanoparticle of an inert solid (commonly gold), which is then "shot" directly into the target cell's nucleus.[27]
Magnetofection, or magnet-assisted transfection, is a transfection method that uses magnetic force to deliver DNA into target cells. Nucleic acids are first associated with magnetic nanoparticles. Then, application of magnetic force drives the nucleic acid particle complexes towards and into the target cells, where the cargo is released.[28]
Impalefection is carried out by impaling cells by elongated nanostructures and arrays of such nanostructures such as carbon nanofibers or silicon nanowires that have been functionalized with plasmid DNA.
Another particle-based method of transfection is known as particle bombardment. The nucleic acid is delivered through membrane penetration at a high velocity, usually connected to microprojectiles.[2]
Other (and hybrid) methods[edit]
Other methods of transfection include nucleofection, which has proved very efficient in transfection of the THP-1 cell line, creating a viable cell line that was able to be differentiated into mature macrophages,[29] and heat shock.
DNA can also be introduced into cells using viruses as a carrier. In such cases, the technique is called transduction, and the cells are said to be transduced. Adenoviral vectors can be useful for viral transfection methods because they can transfer genes into a wide variety of human cells and have high transfer rates.[2] Lentiviral vectors are also helpful due to their ability to transduce cells not currently undergoing mitosis.
Stable and transient transfection differ in their long term effects on a cell; a stably-transfected cell will continuously express transfected DNA and pass it on to daughter cells, while a transiently-transfected cell will express transfected DNA for a short amount of time and not pass it on to daughter cells.
For some applications of transfection, it is sufficient if the transfected genetic material is only transiently expressed. Since the DNA introduced in the transfection process is usually not integrated into the nuclear genome, the foreign DNA will be diluted through mitosis or degraded.[30] Cell lines expressing the Epstein–Barr virus (EBV) nuclear antigen 1 (EBNA1) or the SV40 large-T antigen, allow episomal amplification of plasmids containing the viral EBV (293E) or SV40 (293T) origins of replication, greatly reducing the rate of dilution.[31]
If it is desired that the transfected gene actually remain in the genome of the cell and its daughter cells, a stable transfection must occur. To accomplish this, a marker gene is co-transfected, which gives the cell some selectable advantage, such as resistance towards a certain toxin. Some (very few) of the transfected cells will, by chance, have integrated the foreign genetic material into their genome. If the toxin is then added to the cell culture, only those few cells with the marker gene integrated into their genomes will be able to proliferate, while other cells will die. After applying this selective stress (selection pressure) for some time, only the cells with a stable transfection remain and can be cultivated further.[32]
Common agents for selecting stable transfection are:
Main article: RNA transfection
RNA can also be transfected into cells to transiently express its coded protein, or to study RNA decay kinetics. RNA transfection is often used in primary cells that do not divide.
siRNAs can also be transfected to achieve RNA silencing (i.e. loss of RNA and protein from the targeted gene). This has become a major application in research to achieve "knock-down" of proteins of interests (e.g. Endothelin-1[33]) with potential applications in gene therapy. Limitation of the silencing approach are the toxicity of the transfection for cells and potential "off-target" effects on the expression of other genes/proteins.
^ Transfection at the US National Library of Medicine Medical Subject Headings (MeSH)
^
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^ Transduction, Genetic at the US National Library of Medicine Medical Subject Headings (MeSH)
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^ Sapra, Rachit; Verma, Ram P.; Maurya, Govind P.; Dhawan, Sameer; Babu, Jisha; Haridas, V. (2019-11-13). "Designer Peptide and Protein Dendrimers: A Cross-Sectional Analysis". Chemical Reviews. 119 (21): 11391–11441. doi:10.1021/acs.chemrev.9b00153. ISSN 0009-2665. PMID 31556597. S2CID 203435702.
^ Heitz, Marc; Javor, Sacha; Darbre, Tamis; Reymond, Jean-Louis (2019-08-21). "Stereoselective pH Responsive Peptide Dendrimers for siRNA Transfection". Bioconjugate Chemistry. 30 (8): 2165–2182. doi:10.1021/acs.bioconjchem.9b00403. ISSN 1043-1802. PMID 31398014. S2CID 199519310.
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Scholia has a profile for transfection (Q1429031).
Transfection at the US National Library of Medicine Medical Subject Headings (MeSH)
Biology Research Resource — Articles and Forums about Transfection
Research in optical transfection at the University of St Andrews
See also: RNA vaccine and Transfection
RNA transfection is the process of deliberately introducing RNA into a living cell. RNA can be purified from cells after lysis or synthesized from free nucleotides either chemically, or enzymatically using an RNA polymerase to transcribe a DNA template. As with DNA, RNA can be delivered to cells by a variety of means including microinjection, electroporation, and lipid-mediated transfection. If the RNA encodes a protein, transfected cells may translate the RNA into the encoded protein.[1] If the RNA is a regulatory RNA (such as a miRNA), the RNA may cause other changes in the cell (such as RNAi-mediated knockdown).
Encapsulating the RNA molecule in lipid nanoparticles was a breakthrough for producing viable RNA vaccines, solving a number of key technical barriers in delivering the RNA molecule into the human cell.[2][3]
Contents
RNA molecules shorter than about 25nt (nucleotides) largely evade detection by the innate immune system, which is triggered by longer RNA molecules. Most cells of the body express proteins of the innate immune system, and upon exposure to exogenous long RNA molecules, these proteins initiate signaling cascades that result in inflammation. This inflammation hypersensitizes the exposed cell and nearby cells to subsequent exposure. As a result, while a cell can be repeatedly transfected with short RNA with few non-specific effects, repeatedly transfecting cells with even a small amount of long RNA can cause cell death unless measures are taken to suppress or evade the innate immune system (see "Long-RNA transfection" below).
Short-RNA transfection is routinely used in biological research to knock down the expression of a protein of interest (using siRNA) or to express or block the activity of a miRNA (using short RNA that acts independently of the cell's RNAi machinery, and therefore is not referred to as siRNA). While DNA-based vectors (viruses, plasmids) that encode a short RNA molecule can also be used, short-RNA transfection does not risk modification of the cell's DNA, a characteristic that has led to the development of short RNA as a new class of macromolecular drugs.[4]
Long-RNA transfection is the process of deliberately introducing RNA molecules longer than about 25nt into living cells. A distinction is made between short- and long-RNA transfection because exogenous long RNA molecules elicit an innate immune response in cells that can cause a variety of nonspecific effects including translation block, cell-cycle arrest, and apoptosis.
The innate immune system has evolved to protect against infection by detecting pathogen-associated molecular patterns (PAMPs), and triggering a complex set of responses collectively known as “inflammation”. Many cells express specific pattern recognition receptors (PRRs) for exogenous RNA including toll-like receptor 3,7,8 (TLR3, TLR7, TLR8),[5][6][7][8] the RNA helicase RIG1 (RARRES3),[9] protein kinase R (PKR, a.k.a. EIF2AK2),[10][11] members of the oligoadenylate synthetase family of proteins (OAS1, OAS2, OAS3), and others. All of these proteins can specifically bind to exogenous RNA molecules and trigger an immune response. The specific chemical, structural or other characteristics of long RNA molecules that are required for recognition by PRRs remain largely unknown despite intense study. At any given time, a typical mammalian cell may contain several hundred thousand mRNA and other, regulatory long RNA molecules. How cells distinguish exogenous long RNA from the large amount of endogenous long RNA is an important open question in cell biology. Several reports suggest that phosphorylation of the 5'-end of a long RNA molecule can influence its immunogenicity, and specifically that 5'-triphosphate RNA, which can be produced during viral infection, is more immunogenic than 5'-diphosphate RNA, 5'-monophosphate RNA or RNA containing no 5' phosphate.[12][13][14][15][16][17] However, in vitro-transcribed (ivT) long RNA containing a 7-methylguanosine cap (present in eukaryotic mRNA) is also highly immunogenic despite having no 5' phosphate,[18] suggesting that characteristics other than 5'-phosphorylation can influence the immunogenicity of an RNA molecule.
Eukaryotic mRNA contains chemically modified nucleotides such as N6-methyladenosine, 5-methylcytidine, and 2'-O-methylated nucleotides. Although only a very small number of these modified nucleotides are present in a typical mRNA molecule, they may help prevent mRNA from activating the innate immune system by disrupting secondary structure that would resemble double-stranded RNA (dsRNA),[19][7] a type of RNA thought to be present in cells only during viral infection. The immunogenicity of long RNA has been used to study both innate and adaptive immunity.
Inhibiting only three proteins, interferon-β, STAT2, and EIF2AK2 is sufficient to rescue human fibroblasts from the cell death caused by frequent transfection with long, protein-encoding RNA.[18] Inhibiting interferon signaling disrupts the positive-feedback loop that normally hypersensitizes cells exposed to exogenous long RNA. Researchers have recently used this technique to express reprogramming proteins in primary human fibroblasts.[20]
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a b Angel M, Yanik MF (2010). "Innate Immune Suppression Enables Frequent Transfection with RNA Encoding Reprogramming Proteins". PLOS ONE. 5 (7): e11756. Bibcode:2010PLoSO...511756A. doi:10.1371/journal.pone.0011756. PMC 2909252. PMID 20668695.
^ Herb M, Farid A, Gluschko A, Krönke M, Schramm M (November 2019). "Highly Efficient Transfection of Primary Macrophages with In Vitro Transcribed mRNA". Journal of Visualized Experiments (153). doi:10.3791/60143. PMID 31762462.
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Scholia has a profile for RNA transfection (Q7277187).
DNA and RNA Transfection, Nature (2020}
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2015, Pages 239-252
Chapter 11 - Gene Delivery Strategies
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Abstract
The previous chapter discussed methods for making and manipulating DNA constructs using recombinant DNA technology. Of course, the ultimate goal of creating a DNA construct is to deliver the manipulated sequence into living cells so that the endogenous cellular machinery can transcribe and translate the sequence into functional proteins. A neuroscientist might want to deliver a DNA sequence into a population of cells in culture, in a brain slice, or in the brain of a living animal. The purpose of this chapter is to survey the common methods of delivering recombinant DNA into cells in each of these environments.
Chapter 11
Gene Delivery Strategies
After Reading This Chapter, You Should be Able to:
l Describe common methods of delivering recombinant DNA into cells
l Compare and contrast methods of DNA delivery in various in vitro and in vivo preparations
Techniques Covered:
l Physical gene delivery: microinjection, electroporation, biolistics
l Chemical gene delivery: calcium phosphate transfection, lipid-based transfection
l Viral gene delivery: adenovirus, adeno-associated virus (AAV), lentivirus, herpes simplex virus (HSV), canine adenovirus (CAV)
The previous chapter discussed methods for making and manipulating DNA constructs using recombinant DNA technology. Of course, the ultimate goal of creating a DNA construct is to deliver the manipulated sequence into living cells so that the endogenous cellular machinery can transcribe and translate the sequence into functional proteins. A neuroscientist might want to deliver a DNA sequence into a population of cells in culture, in a brain slice, or in the brain of a living animal. The purpose of this chapter is to survey the common methods of delivering recombinant DNA into cells in each of these environments.
There are three general categories of DNA delivery: physical, chemical, and viral. Transfection refers to nonviral methods of delivering DNA to cells, including physical and chemical methods. Infection refers to viral DNA delivery, in which viruses attach themselves to cells and inject their DNA cargo. Each method has its own relative advantages and disadvantages that make it particularly well-suited for delivering DNA in different experimental contexts (Table 11.1). Major factors that dictate the choice of DNA delivery method include cellular environment, cell type, and experimental goals. Additionally, these DNA delivery methods vary in terms of the number of cells they affect, levels of gene expression they induce, and the length of gene expression over time.
PHYSICAL GENE DELIVERY
Physical gene delivery methods deliver DNA into a cell by physically penetrating the cell membrane with force. These methods are often highly efficient
Guide to Research Techniques in Neuroscience. http://dx.doi.org/10.1016/B978-0-12-800511-8.00011-3
Copyright © 2015 Elsevier Inc. All rights reserved.
239
(transfected cells express high levels of the delivered gene) and can be used with any cell type. However, they can sometimes disrupt the integrity of the cell membrane, traumatizing the cell and leading to its death. Therefore, care must be taken in moderating the amount of force applied to deliver DNA to cells. Three common physical methods include microinjection, electroporation, and biolistics.
Microinjection
A scientist can microinject a solution containing DNA into a cell by piercing the cell membrane with a small glass needle and then applying pressure (Figure 11.1). If the scientist uses an extremely thin needle and withdraws the needle carefully, the cell membrane remains intact, and the cell has an opportunity to incorporate the injected DNA into its genome. This process requires a microscope to view the living cell, as well as fine micromanipulators to precisely place the needle adjacent to the cell membrane. Each cell must be injected one at a time (in contrast to other methods that deliver DNA to millions of cells at once), making microinjection laborious and low throughput. This method requires technical skill and is therefore usually performed by trained technicians or laboratory personnel who use these techniques regularly. Microinjection is rarely used to transfect neurons because their small shape and sensitivity makes them more difficult to inject than other cell types.
TABLE 11.1 Categories of Gene Delivery Strategies
Method
Advantages
Disadvantages
Physical
High-efficiency gene transfer
No limitations on construct size
No cell type dependency
Low throughput
Requires specialized equipment
Can physically harm cells
Chemical
High efficiency in vitro
No limitations on construct size
Relatively easy to perform
Rapid
High throughput
Low immunogenicity
Limited in vivo applications
Efficiency depends on cell type
Can be toxic to cells
Transient expression
Viral
High-efficiency gene transfer
Cell-specific targeting is possible
Long-term expression
Can be used in vitro and in vivo
Complex cloning required
More expensive than transfection methods
Safety concerns regarding production of infectious viruses in humans
May provoke immune response
Laborious preparation
Limited construct size
DNA microinjection is the method used to make transgenic animals. To make a transgenic mouse, a scientist microinjects the transgenic DNA construct into newly fertilized mouse eggs. In some of the new eggs, the transgene randomly incorporates into the mouse genome. These cells are injected into a female mouse to carry the egg to gestation. The full details of creating transgenic mice, as well as other transgenic animals, are discussed in Chapter 12.
Electroporation
Electroporation is the process of using an electric pulse to transfect cells with DNA (Figure 11.2). Applying an electric field to cells is thought to induce temporary pores in the cell membrane, allowing the cell to take up DNA sequences. The electric field also drives negatively charged DNA strands away from the cathode (negative end) toward the anode (positive end) of an electric field. Therefore, an electric pulse causes some of the DNA to enter the cell. After the electric field is switched off, the cell membrane reseals, trapping some of the electroporated DNA within the cell.
Electroporation can be used to efficiently deliver DNA to cells for in vitro conditions using special containers with electrical contacts. In a process known as ex vivo electroporation, a scientist removes a brain from an animal, applies electroporation techniques, and then sections the brain for slice cultures.
In neuroscience research, electroporation has been particularly useful for in utero preparations (Figure 11.3). In utero electroporation surgeries begin by exposing the embryonic pups of a pregnant animal, usually a rodent. DNA is injected into the pups’ ventricles, and electrode paddles direct the uptake of the injected DNA construct(s) toward the anode and away from the cathode. The pups are placed back inside the mother, who is sutured and allowed to recover. Days later, pups are removed or the mother gives birth, and the neurons of the offspring express the introduced DNA construct. Similar methods can be used in the chicken model system in a process called in ovo electroporation. These methods are useful for delivering genes to brain regions adjacent to the
FIGURE 11.1 DNA microinjection. An extremely thin glass needle punctures the plasma membrane, allowing DNA to be injected into the cell.
Gene Delivery Strategies Chapter | 11 241
242 Guide to Research Techniques in Neuroscience nervous system’s fluid-filled ventricles, as the DNA-containing solution must be injected into the ventricle. Thus, they have primarily been used in studies of chick spinal cord and rodent cerebral cortex. A scientist can control both spatial and temporal specificity by controlling the position of the electrode paddles and the timing of electroporation. For example, a scientist can target gene transfection to distinct cortical layers in the mammalian cerebral cortex by electroporating at specific embryonic time periods.
Electroporation has numerous advantages, such as the ability to transfect cells in many different in vivo and in vitro environments. Furthermore, investigators can vary expression levels of a transfected gene by varying the strength and pattern of the electric field pulses.
FIGURE 11.2 Electroporation. (A) Cells are placed in a special chamber with a solution containing the DNA to be transfected. (B) Applying an electric field drives the negatively charged DNA strands away from the cathode and toward the anode. (C) After the electric field is switched off, the cell membrane reseals, and some DNA remains in the cell.
Gene Delivery Strategies Chapter | 11 243 Biolistics
Biolistics, short for “biological ballistics” and also known as particle-mediated gene transfer, is the method of directly shooting DNA fragments into cells using a device called a gene gun.
To use a gene gun, a scientist first mixes a DNA construct with particles of a heavy metal, usually tungsten or gold. These fine particles stick to the negatively charged DNA. The DNA/metal particles are loaded onto one side of a plastic bullet (Figure 11.4). A pressurized gas, usually helium, provides the force for the gun. Gas pressure builds up until a rupture disk breaks, driving the plastic bullet down a shaft. The plastic bullet is abruptly stopped at the end of the shaft, but the DNA/metal particles emerge from the gun with great speed and force. If the gun is aimed at biological tissue, some of the metal particles will penetrate the cell membranes and deliver DNA constructs to cells.
A neuroscientist can use biolistic technology to cause efficient gene expression in neurons. This technology can produce a dispersed transfection pattern, similar to a Golgi stain, in which only individual cells receive the foreign DNA in a background of untransfected cells. Another advantage to using biolistic technology is that a gene gun can deliver DNA through relatively thick tissue, such as a tissue slice in culture. Therefore, it is possible to transfect cells within a slice that would be difficult to target using other gene delivery methods. This technique has not yet been successful in transfecting mammalian neurons in vivo, although it has been used in vivo to transfect liver and skin cells. The main disadvantage of using this technology is that it may cause physical damage to cells. Optimization is required to limit the amount of tissue damage caused by the force of impact of the projectiles.
+ _ Pregnant rodent Developing embryos Injection of DNA construct Application of electric field
FIGURE 11.3 In utero electroporation. A scientist performs a surgical procedure on a pregnant female, exposing the embryonic pups. DNA is injected into the pups’ ventricular system, and paddles generate electric field pulses to introduce DNA into the cells lining the ventricles.
244 Guide to Research Techniques in Neuroscience CHEMICAL GENE DELIVERY
Chemical gene delivery is the process by which a scientist uses a chemical reaction to deliver DNA into cells. Negatively charged DNA can form macromolecular complexes with positively charged chemicals. These complexes can then interact with a cell’s membrane, promoting uptake through endocytosis or fusion. Chemical gene delivery strategies are useful in that they are incredibly high-throughput and often simple to perform. However, they are generally inefficient for in vivo delivery and therefore mainly used for cell culture experiments (Chapter 14). Expression of the target gene is usually transient, lasting for days to weeks depending on the cell type. Common chemical gene delivery strategies include calcium phosphate transfection and lipid transfection.
Calcium Phosphate Transfection
The calcium phosphate method of DNA transfection is one of the simplest and least expensive methods of chemical gene delivery (Figure 11.5). This method requires two chemical solutions: calcium chloride, which serves as a source of
Pressurized gas Rupture disk Bullet DNA-coated metal particles Filter screen Gas breaks rupture disk, driving bullet down the shaft Bullet blocked by filter screen, DNA-coated metal particles emerge Gun aimed at biological specimen
FIGURE 11.4 A gene gun. DNA-coated metal particles are placed on the front end of a bullet. High-pressured gas drives the bullet down a shaft. At the end of the shaft, the bullet is blocked, but the DNA-coated particles emerge with great speed and force.
Gene Delivery Strategies Chapter | 11 245 calcium ions, and HEPES buffered saline (HBS), which serves as a source of phosphate ions. First, the calcium chloride solution is mixed with the DNA to be transfected, and then the HBS is added. When the two solutions are combined, the positively charged calcium ions and negatively charged phosphate ions form a fine precipitate. The calcium ions also cause the DNA to precipitate out of solution. After a few minutes, the solution with precipitate is directly added to cells in culture. By a process that is not entirely understood, the cells take up some of the precipitate, and with it, the DNA. It is thought that the DNA precipitate enters cells by endocytosis, but the exact mechanism remains a mystery. This method works best for a cell monolayer so that the DNA precipitate covers the cells evenly.
The advantages to using the calcium phosphate method are that it is relatively easy, reliable, and cheap. It is useful for transient expression or creating stable cell lines from immortalized cells (Chapter 14). The main disadvantage is that transfection efficiencies are low in neurons (1–3%), and this method does not work at all for transfecting neurons in intact tissue. Some immortalized cell
Solution with DNA and calcium chloride HEPES buffered saline solution DNA precipitates out of solution Solution with precipitate added to cells Ca2+ Ca2+ Ca2+ Ca2+ Ca2+ Ca2+ Ca 2+ Ca2+ Ca2+ Ca2+ Ca2+ Ca2+
FIGURE 11.5 Calcium phosphate transfection. A scientist mixes DNA, calcium chloride, and HEPES-buffered saline. The chemical reaction forms a DNA-calcium phosphate precipitate that is able to pass through the cell membrane and deliver DNA to the nucleus.
246 Guide to Research Techniques in Neuroscience lines, such as HEK 293T and HeLa cells (Chapter 14), exhibit high transfection efficiencies (90–100%), which is one reason for their ubiquitous use in the life sciences.
Lipid Transfection
Lipofection, also known as “lipid transfection” or “liposome-based transfection,” uses a lipid complex to deliver DNA to cells. Lipids are a broad class of fat-soluble biomolecules, such as fats, oils, and waxes. The cell membranes of animal cells are composed of a bilayer of phospholipids with hydrophilic surfaces facing the cytoplasm and extracellular environment (Figure 11.6A). Lipofection technology uses tiny vesicular structures called liposomes that have the same composition as the cell membrane (Figure 11.6B). A scientist performs a simple reaction that forms a liposome around the DNA sequence to be transfected (Figure 11.6C). Depending on the liposome and cell type, the liposome can be endocytosed (Figure 11.6D) or directly fuse with the cell membrane to release the DNA construct into cells (Figure 11.6E).
The advantage to lipofection is that it works in many cell types, including cultured neurons. Commercially available kits allow transfection reactions to be performed within 30 min and gene expression to be assayed within hours.
FIGURE 11.6 Lipofection. (A) The cell membrane is composed of a lipid bilayer, with a hydrophobic interior and hydrophilic exterior. (B) Liposomes are also composed of a lipid bilayer arranged as a spherical shell. (C) A scientist performs a brief reaction that allows liposomes to form around DNA. (D) Cells in culture can endocytose the liposome, digesting it within vesicles to release DNA. (E) Alternatively, liposomes can directly fuse with the plasma membrane, directly releasing DNA into cells.
Gene Delivery Strategies Chapter | 11 247 However, like the calcium phosphate method, lipofection is almost exclusively used in cell culture experiments.
VIRAL GENE DELIVERY
Viral gene delivery uses one of many available viral vectors to deliver DNA to cells in vitro or in vivo. A virus can be thought of as a tiny molecular machine whose entire purpose is to attach to cells and inject genetic material. In nature, this genetic material encodes the proteins necessary to make more virus, so the infected cell essentially becomes a virus-making factory. In the laboratory, many viruses have been manipulated so that they can no longer replicate inside a host cell on their own. Additionally, the coding region of viral DNA is exchanged with a gene of interest to make the virus deliver a transgene to a cell without forcing that cell to produce more virus particles. This process of using a nonreplicating viral vector to deliver foreign DNA into a cell is called transduction.
Scientists can either produce virus in their own laboratories or outsource viral production commercially (Figure 11.7). To begin, a scientist uses recombinant DNA technology (Chapter 10) to place a DNA sequence of interest into a plasmid containing the necessary sequences to incorporate into a virus particle. Note that this plasmid is not the same as a virus itself but is simply a piece of DNA with the necessary sequences to incorporate into a virus particle, provided the other necessary proteins are present. To produce a batch of virus, cultured cells are used to produce all the necessary viral components. A scientist transfects these cells with the recombinant plasmid, as well as additional DNA sequences coding for the necessary viral proteins. Because viral DNA has been engineered so that it cannot replicate more virus on its
Transfect cells with plasmids encoding viral particles After 2 days, collect viral-containing supernatant Centrifuge to collect viral pelletResuspend pellet in buffer
FIGURE 11.7 Virus production. A scientist transfects cultured cells with DNA encoding the necessary proteins to make virus. Over 2–3 days, the cells produce virus and release it into the culture medium. The medium is collected and centrifuged to collect a viral pellet. The pellet is resuspended in buffer and frozen until use.
248 Guide to Research Techniques in Neuroscience own, these viral proteins are necessary to produce functional, infectious viral particles. Immortalized cell lines, such as HEK 293T cells, are the best cells to serve as virus packaging cells because they grow quickly, are transfected relatively easily, and can produce large amounts of virus. One to two days after transfection, the packaging cells produce many virus particles and release them into the extracellular medium. The final step of virus production is to collect the medium, filter out cell debris, and centrifuge the medium to collect the viral pellet. The pellet is dissolved in a buffer and, depending on the needs of the investigator, either used immediately to infect the target cells of interest or frozen until needed.
Viral infections are robust, highly efficient, and can lead to long-term expression. Viruses have been used to deliver genes to cultured cells, brain slices, tissue explants, and brain regions in vivo. They are the tools of choice to deliver genes into the brains of rodents, as most other methods of gene delivery are incapable of working efficiently in vivo. However, there are also disadvantages and limitations to using viral gene delivery. The infected cells, especially cells in vivo, may not begin expressing the transduced gene until 7–14 days after exposure to the virus. Also, the size of the viral vector restricts the size of the DNA construct that a scientist can transduce. Finally, scientists must take great care and safety when using viruses because it is possible for scientists to accidentally infect themselves.
There are a variety of viral vectors that are useful to neuroscientists (Table 11.2). They vary in terms of infection efficiency, expression levels, duration of expression, time to start of expression, host cell toxicity, and host cell preference. Adeno-associated virus and lentivirus are the most commonly used viral vectors due to long-term expression of transgenes and low toxicity in neurons.
Adenovirus
Adenovirus can infect both dividing and postmitotic cells in a broad range of species and cell types. It can carry DNA constructs that are 7.5–30 kb (kilobases) long. The DNA does not integrate into the genome, so expression is transient, lasting weeks or months. It is useful for high-level transient expression in neurons, but it can often cause an inflammatory response and cell death.
Canine Adenovirus
Canine adenovirus (CAV) is a form of adenovirus that is highly infectious in dogs. This virus has a high carrying capacity of 7.5–30 kb and is used in neuroscience research because of its retrograde properties. Upon injection into the brain, CAV can be taken up by the presynaptic membrane and retrogradely transported to cell soma. Therefore, this viral vector is useful for targeting neurons based on their connectivity.
Herpes Simplex Virus
Herpes simplex virus (HSV) is a neurotropic virus that naturally targets neurons as host cells. It has a very large genome compared to other viruses (100–200 kb) that allows delivery of long DNA sequences. While the DNA does
250 Guide to Research Techniques in Neuroscience not integrate into the host genome, it exists stably in the nucleus to produce long-term (months to years) expression. The disadvantages are that it can be difficult to engineer HSV because of its large size, and it can be toxic to cells. There are also safety concerns because it is highly infectious.
Adeno-Associated Virus
Adeno-associated virus (AAV) is naturally replication deficient, requiring a helper virus to replicate, so it is safer to use than other viral strains. AAV can infect both dividing and postmitotic cells, but, unlike adenovirus, does integrate into the host genome, permitting long-term gene expression. AAV also seems to be less toxic to neurons than adenovirus. However, AAV production is labor-intensive and the carrying capacity is much less than adenovirus, about 5 kb.
Lentivirus
Lentivirus belongs to a class of virus called retrovirus that has an RNA genome rather than DNA. To produce functional gene products, the virus also contains the enzyme reverse transcriptase, which produces cDNA from the RNA template (Chapter 10). When a cell endocytoses a lentivirus particle, the RNA is released and reverse transcriptase produces cDNA. The DNA migrates to the nucleus, where it integrates into the host genome.
Most retroviruses only infect dividing cells, making them useful for studying neuronal development and cell fate. However, lentivirus is capable of infecting both dividing and postmitotic cells (e.g. neurons) and is therefore widely used in neuroscience experiments. Lentivirus is based on the human immunodeficiency virus and has an 8-kb carrying capacity. Because the DNA integrates into the genome, lentivirus delivery leads to long-term expression.
CONCLUSION
This chapter has surveyed common methods used to deliver DNA sequences into cells. A scientist chooses one method over another based on a number of factors, such as the cell type and the cell’s environment, as well as the specific goals of the experiment (Table 11.3). For cell culture experiments, chemical, electroporation, or viral gene delivery strategies all work well. For in vivo DNA delivery to adult animals, viral gene delivery is the most efficient option. Now that we have surveyed these methods, the next two chapters will focus on methods of manipulating the genomes of living organisms.
SUGGESTED READING AND REFERENCES
Books
Heiser, W. C. (Ed.). (2010a). Gene delivery to mammalian cells. Volume 1: Nonviral gene transfer techniques. New York, NY: Humana Press.
Heiser, W. C. (Ed.). (2010a). Gene delivery to mammalian cells. Volume 2: Viral gene transfer techniques. New York, NY: Humana Press.
Twyman, R. M. (2005). Gene transfer to animal cells. Abingdon, Oxon, UK: Garland Science/BIOS Scientific Publishers.
Review Articles
Bonetta, L. (2005). The inside scoop—evaluating gene delivery methods. Nature Methods, 2, 875–883.
Callaway, E. M. (2008). Transneuronal circuit tracing with neurotropic viruses. Current Opinion in Neurobiology, 18, 617–623.
Luo, L., Callaway, E. M., & Svoboda, K. (2008). Genetic dissection of neural circuits. Neuron, 57, 634–660.
Washbourne, P., & McAllister, A. K. (2002). Techniques for gene transfer into neurons. Current Opinion in Neurobiology, 12, 566–573.
Primary Research Articles—Interesting Examples from the Literature
Carter, M. E., Soden, M. E., Zweifel, L. S., & Palmiter, R. D. (2013). Genetic identification of a neural circuit that suppresses appetite. Nature, 503, 111–114.
Chen, J. L., Carta, S., Soldado-Magraner, J., Schneider, B. L., & Helmchen, F. (2013). Behaviour-dependent recruitment of long-range projection neurons in somatosensory cortex. Nature, 499, 336–340.
Krashes, M. J., Shah, B. P., Madara, J. C., Olson, D. P., Strochlic, D. E., et al. (2014). An excitatory paraventricular nucleus to AgRP neuron circuit that drives hunger. Nature, 507, 238–242.
Pekarik, V., Bourikas, D., Miglino, N., Joset, P., Preiswerk, S., & Stoeckli, E. T. (2003). Screening for gene function in chicken embryo using RNAi and electroporation. Nature Biotechnology, 21, 93–96.
Tsai, H. C., Zhang, F., Adamantidis, A., Stuber, G. D., Bonci, A., de Lecea, L., et al. (2009). Phasic firing in dopaminergic neurons is sufficient for behavioral conditioning. Science, 324, 1080–1084.
Wilson, S. P., Yeomans, D. C., Bender, M. A., Lu, Y., Goins, W. F., & Glorioso, J. C. (1999). Antihyperalgesic effects of infection with a preproenkephalin-encoding herpes virus. Proceedings of the National Academy of Sciences of the United States of America, 96, 3211–3216.
Protocols
Lappe-Siefke, C., Maas, C., & Kneussel, M. (2008). Microinjection into cultured hippocampal neurons: a straightforward approach for controlled cellular delivery of nucleic acids, peptides and antibodies. Journal of Neuroscience Methods, 175, 88–95.
O’Brien, J., & Lummis, S. C. (2004). Biolistic and diolistic transfection: using the gene gun to deliver DNA and lipophilic dyes into mammalian cells. Methods, 33, 121–125.
Walantus, W., Castaneda, D., Elias, L., & Kriegstein, A. (2007). In utero intraventricular injection and electroporation of E15 mouse embryos. Journal of Visualized Experiments, 6. http://dx.doi. org/10.3791/239. http://www.jove.com/index/details.stp?id=239.
Websites
University of North Carolina Vector Core: http://www.med.unc.edu/genetherapy/vectorcore.
University of Pennsylvania Vector Core: http://www.med.upenn.edu/gtp/vectorcore/.