Introduction
The ability to express foreign or specific genes in cultured cells is a powerful tool for studying gene expression. This approach enables the monitoring of transcriptional and translational activities of introduced DNA, facilitates the functional analysis of specific genes, allows for protein production and characterisation, and supports the exploration of regulatory mechanisms in a controlled environment. Expressing genes also allows the development of model systems for disease research, the production of recombinant proteins for therapeutic or industrial use, investigation of gene interactions, and the testing of gene-based therapies in a controlled environment.
In this module, we will look at the types of vectors that are available for introducing specific or foreign genes into cells.
Outline:
1. How to create effective vectors (choosing and designing them for Gene delivery).
2. Vectors and how they're used in different Gene Delivery Systems
3. A look at different systems and different approaches to gene delivery
What is gene delivery and why would you want to perform such an experiment?
Gene delivery encompasses a wide range of different applications and different approaches. Fundamentally, gene delivery is pretty straightforward: it is the taking of a foreign or specific piece of genetic material and delivering it to your host cells. These kinds of operations are not only utilised for research in the life sciences and for clinical advancement, but are also very influential in a wide range of other industries.
Gene delivery specifically in the Life Sciences, allows us to perform a range of studies including:
a. Observing endogenous gene expression
b. Modulating pathways
c. Editing a host organism's genome.
The options for different experimental approaches are vast and can be daunting. Not only do we have a wide range of options for what we can do with the genetic material once it gets into cells, we also have a wide array of options for how to get that genetic material into the cells in the first place.
At this point we must get familiar with the concept of a vector. Vectors at the core are essentially a mode of transport for getting our DNA or RNA into our host cell. Vectors take a variety of different forms and include:
a. Viruses
b. Plasmids and
c. RNA
Whether you choose viruses, plasmids or RNA as your vector, within each category, there are multiple different subtypes. Yet, regardless of what application or delivery system you utilise, it all starts the exact same way: with a plasmid.
The basic components of a plasmid that makes it indispensable as a gene delivery vector are:
a. An origin of replication: this is what allows the plasmid to replicate in bacteria. It is the site on the plasmid that initiates replication of our gene, once the plasmid is introduced into bacteria. There are different origins of replications including pUC ori, which allows high copies of the plasmid (and therefore your gene), to be made.
b. An antibiotic resistance gene: these sites on plasmids offer the ability for the bacteria harbouring such a plasmid, to be resistant to specific antibiotics. A popular antibiotic that the plasmid may offer resistance to is ampicillin. But there are others depending on your host cell, such as blasticidin or pyrimethamine, for those in malaria research. Antibiotic resistance markers allow us to select for and grow only the bacteria that have taken up our plasmid. This assists us in creating numerous copies of the genetic material that we want to deliver to our cells.
The genetic material we want to deliver often takes the form of either a gene, where we have something called an Open Reading Frame (ORF) encoding a protein coding gene, or it can be an RNA. Once we have lots of copies of the genetic material, then we can start thinking about how to get it into our host cells.
Since we are ultimately wanting to express our genetic material in a host cell, we need our plasmid to have additional components that facilitate identification of our introduced genetic material.
Essentially, in our host cells, we want to know which cells have actually taken up this plasmid (or other vector that we introduced). This additional component can either be another antibiotic selection marker, for instance puromycin, or it can be a fluorescent screening marker.
The most popular fluorescent marker is a protein called Enhanced Green Fluorescent Protein (EGFP). This marker allows us to visualise which host cells have the vector.
In order to make the genetic material function, (i.e be transcribed and translated into a protein), once inside the host cell, we need additional plasmid components that target the host cell. These host related components include:
a. A promoter: to drive gene expression
b. A poly-A signal: serves as the transcriptional termination site and addition of a polyA tail to mRNA.
c. A Kozak sequence: (straight after the promoter) that serves as protein translation initiator for the gene that we are driving expression of.
Now that we have our plasmid, it's ready to go into the host cell, where it should be functionally active.
Modes of Transport for our Genetic Material or RNA of Interest into Host Cells.
Viral Vectors
If you want to create a recombinant virus for gene delivery, there are extra components as well as extra plasmids that you have to consider.
a. The plasmid that's carrying your gene or your RNA to be delivered, is called the transfer vector. This is the plasmid that contains the host components and the bacterial components, as well as parts of the viral genome that are needed for delivery.
b. For increased efficiency, we additionally need packaging vectors. A packaging vector carries other components that are needed to create a live virus. These packaging components are often separated over multiple vectors to increase both safety, as well offer tight control over the production of the virus.
This consequently makes viral vectors more complex than the regular plasmid we have discussed. This initial increased work offers advantages when it comes to introducing our genetic material into a host cell. Remember that plasmids (and other non-viral vectors) on their own, are mere nucleic acids. Hence, it's very difficult to get them across a cell membrane into our host cells.
A Close Look at Viral vs. Non-viral Options
For non-viral vectors we have to transfect the genetic material into host cells. This can be done using methods like electroporation or Lipid Nano Particle (LNP) delivery, or micro injection. These delivery methods are referred to as transfection.
This contrasts with the technique for introducing viral vectors, called transduction. With transduction, you simply expose the cells to your recombinant virus, avoiding the need for additional reagents or aids.
Transfection can often be harsher and or more technically difficult than transduction.
Non-viral vectors require greater optimisation or work at the delivery end, but they are much easier to produce and are often better tolerated by cells. Especially if you're putting this in a whole organism and doing in-vivo transfection (you tend to have lower immune reactions). However, in-vivo delivery can be relatively difficult, especially if you're trying to target very specific cells. In such settings, non-viral vectors usually have lower efficiencies.
Conversely, viral vectors offer highly specific and efficient delivery, but they tend to have higher immune reactions, potentially causing toxicity. In addition, they can exhibit longer term off-target effects, depending on how they are designed and how they're used.
In terms of decision making, in general, non-viral vectors are a popular option for easy to transfect cells, while viral vectors are better for in-vivo or for difficult to transfect cells.
Popular Non-viral Vectors
The most commonly used non-viral vectors include:
Regular plasmids
Transposons
RNA
While these tend to be simpler and easier to produce - with lower toxicity and immunogenicity - they have several disadvantages. Namely, they are more complicated to deliver to your host cell and tend to be less efficient than viral methods.
The major differentiator between these three non-viral vectors is the speed and length of effect. Specifically, RNA is already transcribed and so you're going to have very fast and very efficient activity. However, it has lower stability and host cells readily degrade RNA. Consequently, they are much shorter lived.
In contrast, regular plasmids must go through the transcription and or translation process - as it is DNA - so they're going to be much slower to initiate activity. Regular plasmids are simpler and more stable, which means that they're going to have longer lived activity. Additionally, they can generally carry more cargo than RNA. Yet, it is less efficient in many cell types, including non-dividing cells, which RNA tends to be slightly better at targeting.
With regular plasmids, you have the potential for genomic insertion, though the likelihood is low. If insertion is actually what you're looking for, then transposons are a better option.
Transposons are designed for genomic integration and there are a variety of options available, including Piggyback, Tol2 and Sleeping Beauty. The downside to transposons is that you typically require multiple vectors and because these are plasmids they are going to have slightly lower efficiency at transfecting cells. The onset of action is also relatively slow.
Thus, if you're considering non-viral vectors, your choice depends on how fast and how long you want that activity to be. These considerations equally apply to viral vectors.
Popular Viral Vectors
Lentivirus
Adeno-associated Virus (AAV)
Adenovirus
When considering viral vectors, the choices range from those that primarily stably integrate the gene or RNA of interest into your genome, versus those that have more transient expression.
For the viruses that typically do not integrate into the genome, adeno-associated virus and adenovirus, are the most popular options. The difference between these two largely comes down to how big your host components are. That is your promoter, your ORF, and your marker, etc.
If your insert is less than 4.7 kb, adeno-associated virus tends to be the obvious choice. Whereas if the insert is larger (7.5 - 33 kb), then adenovirus is fit for purpose.
AAV has the benefit of very low immunogenicity when used in-vitro. You can also take advantage of various different serotypes: for example, the capsid for the AAV virus can be selected to give you either broad or very specific targeting or tropism.
For stably integrating viruses, lentivirus (6.4 kb) tends to be the go-to. It has a larger carrying capacity and it also has the benefit of very broad tropism, being able to infect both dividing and non-dividing cells. Additionally you can customize your promoter.
Other viruses that can integrate are Moloney Murine Leukaemia Virus (5.5 kb) and Murine Stem Cell Virus (6.1 kb). These tend to be historical viral vectors that are used due to familiarity, especially for hematopoietic stem cells.
Less popular viral vectors that are utilised for specific applications include:
a. Vascular stomatitis virus (VSV): this can be readily pseudotyped to put different proteins on the viral envelope, allowing it to either have specific targeting abilities or mimic other viruses and study viral entry mechanisms.
b. Vaccinia virus (as well as VSV): are excellent options for vaccine development. This is due to its broad tropism or targeting.
c. Herpes Simplex Virus (HSV): is fit for oncolytic studies.
d. Rabies: is ideal for the labelling of neural networks.
Additional considerations for whether to choose a viral or non-viral vector include, which application we are using the vector for. For example, whether we are interested in over-expression, knockdown or knockout. Also whether we're using mice, human, zebra fish, drosophila or plants, impact the type of approach that is suitable.
Designing a Gene Editing Vector
Once we have our vector (and our packaging systems, if using a viral approach), we need to insert something (our gene of interest) into the vector. Whether you're using a standard plasmid or transposon or lentivirus, you're typically going to start with a preformed backbone. The backbone should have the necessary components discussed above.
Focusing on host components, the cargo (the gene of interest and its accessories) would include a promoter. The promoter drives expression of the gene you want.
A Closer Look at Promoters
There are a number of considerations that we have to take into account in choosing a promoter. Firstly, if we are expressing RNA, we have a few different options. In instances where we're trying to get RNA expression outside of cells for in-vitro transcription, a T7 promoter with T7 polymerases is going to be the default option.
In cases where you desire to have your cells perform an action, for example, you deliver the vector to your cells and you want to create a small RNA or an shRNA, or guide RNA (gRNA), you have a couple of different options. The most popular of these is to use a Pol III promoter. The Pol III promoter is associated with the production of small RNAs. The most common of this type is called U6 and it drives systemic expression of shRNA. The caveat is that we can only have one shRNA ubiquitously expressed everywhere with this promoter. The benefit of this promoter is that you are likely to have high efficiency knockdown for the expressed shRNA.
Alternatively, there is another shRNA system that uses a different promoter: a Pol II promoter. The Pol II promoter is associated with the production of mRNA leading to a functional protein. One such system is called the miR30 system - you can have multiple shRNAs. You can also customise your promoter. The disadvantage with this system is that you have reduced efficiency of the knockdown.
Finally if you want to produce mRNA that results in the production of a protein in cells, you likewise require a Pol II promoter. In such scenarios, other considerations including the location and time of expression of the transgene must be tackled. If we want system wide expression, we have a variety of different ubiquitous promoters to choose from. It may be instinctive to think that the strongest promoter would be the best. However, this may not always be advisable. Consider a scenario where you have a protein that is efficiently translated, coupling it to a strong promoter may overwhelm the cell. Or if you have a toxic gene, in such circumstances, a strong promoter is deleterious.
Also, you have to consider which cells you are going to target with a ubiquitous promoter. If you're trying to target every cell in the entire body, (for instance, in an animal model), you want to avoid a Cytomegalovirus (CMV) promoter. This is because CMV promoters tend to be silenced in some cell types, when used in-vivo.
Finally, there are some considerations that are peculiar to lentivirus. In experiments with lentiviral vectors, where various ubiquitous promoters were tested, it was discovered that the CMV derived synthetic promoter, CAG, tends to be associated with lower titres and lower efficiency. Presumably because of a high GC content. Additionally, it's best to avoid using the same promoter twice. For example, the promoter for your ORF and the promoter for your marker must not be the same. This is due to the possibility for the promoters to recombine, resulting in the loss of all of the genetic material in between those two promoters.
Controlling When and Where a Gene is Expressed
In instances where we're being selective in the location or timing of expression of the transgene, the options are relatively straightforward. You may use a tissue specific promoter or a promoter that is regulated so that it is conditionally expressed.
You may use a recombinase enzyme such as Cre or the yeast transcription factor, Gal4 UAS to control where the gene is expressed. However where you do not have an existing Cre or Gal4 expressing cell line, a tissue specific promoter is suitable. Researchers can take advantage of the tropism of different viruses to enhance targeting. As mentioned earlier, different AAV serotypes can target different types of cells or you can pseudotype different viruses like VSV and lentivirus to enhance that specificity.
You may also control timing by utilising a tetracycline-inducible expression system. Here, you control gene expression with the addition of an activator (doxycycline) or removal of a repressor (doxycycline). Note that such approaches often require optimisation to obtain complete expression or repression. An effective strategy is to combine a repressor with an activator in an inducible system, in order to gain greater control over the expression or repression.
A popular option for regulating the location and time of gene expression, is via the expression of an RNA. For instance, an shRNA or a gRNA for regulation or potentially, genome editing. shRNAs are typically used for knockdown experiments and are relatively accessible because databases exist where we can input our gene of interest and obtain numerous options of 21 nucleotide hairpin structures (shRNAs). The top scoring shRNAs can then be inserted into our vector for validation. It is recommended that at least three shRNAs are tested to ensure that you have convincing validation.
Another option for RNA that can be expressed are gRNAs for CRISPR systems. Here, as with the shRNA option, you can use databases to search for gRNA sequences for your gene of interest. By uploading the gene sequence of interest, you will obtain suggestions of 20 nucleotide gRNAs that are scored according to specificity and the likelihood for on-target efficiency and potential off-target effects. With gRNAs - unlike shRNAs - we must factor in accessibility of our target site by the gRNA-CAS9-complex. We additionally have to ensure that a sequence of nucleotides, known as a Protospacer Adjacent Motif (PAM), is in the vicinity.
Additional Considerations for Designing Expression Vectors
If we wish to express a protein, it's recommended to invest the time to ensure your insert is codon optimized. This involves replacing certain codons in the gene with more commonly used codons, provided that they are synonymous. This ensures that the sequence that you have, corresponds with the most highly used codons in your host organism. This ensures that the translation of the resultant transcript is efficient.
Also, when expressing more than one gene, for example, a pathway specific protein and a selection marker, it is best practice to place the important gene (pathway specific gene) first, and then the marker second. This is based on the observation that the gene that is placed upstream tends to have higher expression levels. Illustratively, if we have a promoter driving the ORF or our RNA, then our next thing that we have to consider is what's downstream of that. If it's nothing, we can insert a PolyA signal and that completes the vector. On the other hand, if we want to express multiple genes, there are various approaches.
Firstly, we can situate the genes we wish to express beside each other, by encoding them as completely separate cassettes. For example, you can have each gene with a different promoter, separated by the polyA tails. However, such an approach is not suitable for lentiviral vectors. This is because when making a lentiviral transfer vector, you cannot have an internal polyA signal. Doing so, dramatically reduces the viral titers. The alternative is to utilise two ORFs that are linked. This results in the production of a single mRNA, that is translated to produce two separate proteins. A popular linker that is commonly used is the so-called Internal Ribosomal Entry Site (IRES). In eukaryotic cells, translation typically starts at the 5' cap of mRNA. However, IRES elements allow ribosomes to bind to mRNA internally, bypassing the need for the 5' cap. This is a good option, particularly if your second ORF is a marker, because the downside here is that your second ORF will be translated at lower efficiency. Note that when using an IRES, you have two separate translation events. Hence, you require a stop codon at the end of that first ORF.
Alternatively you can use a 2A linker. 2A peptides are short amino acid sequences (typically 18-22 residues) that induce ribosomal skipping during translation. With this strategy, the ribosome translates the upstream gene, but upon encountering the 2A peptide sequence, the ribosome pauses. The first product "cleaved" between a glycine and proline residue within the 2A peptide. For this approach you do not include a stop codon, as the same ribosome travels along the entire mRNA. The disadvantage of this approach is that you get additional amino acid residues that are on either side of that cleavage point. Hence, this is not always suitable, if the additional residues might interfere with the functioning of your protein.
The last approach is to perform a fusion of those two proteins. With this approach, you create a single protein with functions of both of those genes. However, depending on the end goal, this approach will need optimisation. For example if we desire to express EGFP with an antibiotic selection marker, such as neomycin, the orientation is critical for the expression of the EGFP. However, should we wish to pair the EGFP with the antibiotic puromycin, we are restricted to using a two-way linker, rather than a fusion protein approach.
Conclusion
The selection of an appropriate vector for gene delivery is a critical decision that impacts the success of your experiment. The choice is shaped by a variety of factors including the type of host cells, the nature of the genetic material being delivered, and the desired outcome of the gene expression.
Viral vectors, with their high efficiency and specificity, are ideal for in vivo applications and difficult-to-transfect cells, while non-viral vectors offer simpler production and lower immunogenicity, making them suitable for certain research contexts.
Bibliography
Wu Y, Kirkman L, Wellems TE. Transformation of Plasmodium falciparum malaria parasites by homologous integration of plasmids that confer resistance to pyrimethamine. Proc Natl Acad Sci U S A. 1996;93(4):1114-1118.
Mamoun C, Gluzman I, Goyard S, Beverley SM, Goldberg DE. A set of independent selectable markers for transfection of the human malaria parasite Plasmodium falciparum. Proc Natl Acad Sci U S A. 1999;96(15):8633-8638.
Nishi T, Shinzawa N, Yuda M, Iwanaga S. Highly efficient CRISPR/Cas9 system in Plasmodium falciparum using Cas9-expressing parasites and a linear donor template. Sci Rep. 2021;11(1):1-10.
Adjalley S, Lee MCS, Fidock D. A method for rapid genetic integration into Plasmodium falciparum utilizing mycobacteriophage Bxb1 integrase. Methods Mol Biol. 2010.
O'Donnell R, Freitas-Junior L, Preiser P, et al. A genetic screen for improved plasmid segregation reveals a role for Rep20 in the interaction of Plasmodium falciparum chromosomes. EMBO J. 2002;21(5):1013-1020.
Epp C, Raskolnikov D, Deitsch K. A regulatable transgene expression system for cultured Plasmodium falciparum parasites. Malar J. 2008;7(1):1-10.
Knuepfer E, Napiorkowska M, van Ooij C, Holder AA. Generating conditional gene knockouts in Plasmodium– a toolkit to produce stable DiCre recombinase-expressing parasite lines using CRISPR/Cas9. Sci Rep. 2017;7:3881.
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