Plasmids Crash Course: Many Hosts, One Plasmid Guest
Plasmids are at the heart of gene delivery experiments, as they can be used to over-express a transgene, knocking a gene out, or modulate gene expression. Often a single plasmid is the workhorse of the experiment. That is a lot of responsibility hanging on this tiny circle of DNA, so there are many important components to consider. Here we’ll review some basics of plasmid components to make sure everyone is accounted for in your experimental design.
Our first post in “My Favorite Building Block” lays the foundation for how plasmids are made and highlighted some of their endless possible applications. These small circles of DNA can be the landing spot for genetic material that you want to introduce, whether this is a transgene to be expressed, CRISPR/Cas9 components, or small non-coding RNAs. Once the genetic material you are interested in introducing is cloned into your plasmid, bacterial hosts engineered to propagate and replicate the plasmid can grow at an exponential rate if grown in the right conditions, leading to a huge number of plasmids grown in culture overnight.
After you produce a large amount of plasmid DNA in your bacterial host, your gene typically needs to be expressed in a different host, e.g. in a mammalian, plant, yeast cell. Additionally, there may be intermediate steps that involve another host, e.g. a virus being produced in packaging cells. Each step and each host must be appropriately accounted for to ensure a successful gene delivery experiment.
Friends to bacteria
In order to propagate a plasmid after initial cloning, it must first be transformed into a bacterial host cell. Bacteria are then grown up in culture, and as they replicate, they also replicate the plasmid. In addition to your chosen genetic material, the plasmid must have basic components to interact with the bacterial host (Figure 1). Basic components you will find in almost any plasmid are (1) an origin of replication (ori, often seen as pUC ori), the initiation site for plasmid DNA replication recognized by the bacteria, and (2) a selection marker, often antibiotic resistance. The pUC ori produces high copy numbers of a plasmid while the selection marker allows for antibiotics to kill all bacteria that are not host to the plasmid.
Figure 1. Bacteria-related plasmid components (blue) necessary for propagating your GOI (purple).
Some specialized plasmids also contain elements that facilitate genomic integration into host cells. PiggyBac, Sleeping Beauty, and Tol2 contain repeated sequences flanking your GOI that serve as recognition sites for transposase. PiggyBac and Tol2 use inverted terminal repeats (ITRs), while Sleeping Beauty uses inverted/direct repeats (IR/DRs).
Friends to viruses
While plasmids are easily transformed into bacterial cells with heat shock, permeabilization, or electroporation, transfecting into other cell types can be more difficult. Often viruses are used to transduce cells in vitro or in vivo for efficient gene delivery. The production of recombinant viruses requires important components on transfer and packaging plasmids. These viral vectors responsible for making a recombinant virus contain 3 elements:
- The genetic material you want to introduce, for instance your GOI.
- Genes necessary to make viral components, especially the capsid and/or envelope.
- For lentivirus, these are env and VSV-G.
- For AAV, these are the Cap and aap genes.
- Genes necessary to package your genetic material into the virus.
- For lentivirus, these are the gag, pol, and rev genes.
- For AAV, this is Rep.
Viral vectors may include other components, including those that integrate your GOI into the host genome. For example, lentivirus as well as retroviruses MMLV and MSCV require long terminal repeats (LTRs) on either side of the GOI which facilitate their integration and transcription. Alternatively, AAV utilizes inverted terminal repeats, which fold over like paper clips, to package its single-stranded DNA genome for episomal transgene expression (Figure 2).
Figure 2. Terminal repeats facilitate genomic integration or efficient introduction of viral genome.
Viral genes must contain a promoter that is recognized by an RNA polymerase. The promoter regions used for virus production are often viral promoters, including Rous sarcoma virus (RSV) and cytomegalovirus (CMV) promoters. The latter is so powerful that it is a commonly used ubiquitous promoter in recombinant gene technologies in both mammalian and non-mammalian systems.
Viral components are separated onto different plasmids to increase the safety of the system and all of the plasmids with the separate components must be present to create a recombinant virus. For example, producing recombinant AAV for transgene expression requires 3 plasmids: one with the AAV capsid and packaging genes, one containing a transgene flanked by ITRs, and one with adenovirus helper genes which facilitate viral replication (Figure 3). As discussed above, all of these vectors must still contain the components for replication and selection in a bacterial host.
Figure 3. Plasmids necessary for production of recombinant AAV, consisting of bacterial components (blue), viral components (green), and the GOI (purple).
Friends to everyone
Cloning your plasmids, propagating them in bacterial cells, and using them to create recombinant viruses is a weighty task that hopefully ends in your genetic material being introduced to your final host cells (if you’re having trouble with that, check out our Viral Packaging services).
Whether you are inducing transgene expression, performing CRISPR gene editing, or testing regulatory functions of an enhancer, your plasmids need to be designed properly to interact with their potential host cells. Often, a ubiquitous promoter like CMV can be applied to facilitate plasmids use in multiple cell types. However, it is important to ensure that your promoter is functional in your host, whether your cells are mammalian, fish, avian, etc.
Downstream of the promoter a variety of regulatory elements and functional signals can be inserted to increase transgene expression or facilitate the expression of multiple transgenes. Linkers (e.g. T2A) can be placed between ORFs to express multiple transgenes off a single transcript. Other popular elements include polyA signals for efficient gene expression and the WHV Posttranscriptional Regulatory element (WPRE) which enhances gene expression (Figure 4).
Figure 4. Plasmids necessary for expression of transgene (ORF) using AAV, consisting of bacterial components (blue), viral components (green), and host gene expression components (purple).
As shown by the number of vector systems available at VectorBuilder, there are a huge number of additional elements that allow for propagation, replication, selection, packaging, and expression in your host cells. These vectors can be designed by working outward: starting with your application, then adding components for delivery, and finally ensuring that components are present for growth or activity in each host.
Plasmid maps can be complex and can seem intimidating. However, by breaking components down into their particular host and the relevant step in gene delivery, you can not only appreciate the importance of each element but also ensure that your plasmid has everything it needs for success in bacteria and beyond. You can explore various vector systems and components in our Guides as well as our Vector Design Studio, and we are always on hand to help from design through to virus packaging.
Bouard D, Alazard-Dany D, Cosset FL. Viral vectors: from virology to transgene expression. Br J Pharmacol. 2009 May;157(2):153-65. doi: 10.1038/bjp.2008.349. PMID: 18776913; PMCID: PMC2629647.
del Solar G, Giraldo R, Ruiz-Echevarría MJ, Espinosa M, Díaz-Orejas R. Replication and control of circular bacterial plasmids. Microbiol Mol Biol Rev. 1998 Jun;62(2):434-64. doi: 10.1128/MMBR.62.2.434-464.1998. PMID: 9618448; PMCID: PMC98921.
Gill DR, Pringle IA, Hyde SC. Progress and prospects: the design and production of plasmid vectors. Gene Ther. 2009 Feb;16(2):165-71. doi: 10.1038/gt.2008.183. Epub 2009 Jan 8. PMID: 19129858.
Matsushita T, Elliger S, Elliger C, Podsakoff G, Villarreal L, Kurtzman GJ, Iwaki Y, Colosi P. Adeno-associated virus vectors can be efficiently produced without helper virus. Gene Ther. 1998 Jul;5(7):938-45. doi: 10.1038/sj.gt.3300680. PMID: 9813665.
Naso MF, Tomkowicz B, Perry WL 3rd, Strohl WR. Adeno-Associated Virus (AAV) as a Vector for Gene Therapy. BioDrugs. 2017 Aug;31(4):317-334. doi: 10.1007/s40259-017-0234-5. PMID: 28669112; PMCID: PMC5548848.