Note: The images and tables below are adapted from VectorBuilder. This post and I have no affiliation with them.
Our genomes hold a counterintuitive truth: their most abundantly transcribed sequences don't code for proteins at all. Despite never being translated, these non-coding RNAs (ncRNAs) fine-tune crucial processes that keep our cells alive and functioning precisely. In recent years, long non-coding RNAs (lncRNAs) have gained significant attention due to continued findings about their biological impacts and potentials as therapeutic targets. While studying them in the lab, we often start with the classic approach: transiently transfecting cells with over-expression plasmids to see what happens when we dial up their levels. It can work, but it's far from perfect. You never quite get every cell on board (goodbye, 100% efficiency dreams), and in longer experiments like a two-week-long soft agar colony formation assay, you're fighting a battle against cell division. With each split, the plasmids get divided between daughter cells, gradually dimming the signal you're trying to study.
Generating cell lines that stably express a lncRNA of interest offers an attractive option to mitigate these issues. One of the most common methods of stable cell line generation is through the use of lentivirus vectors - the focus of this blog post. However, there are some key considerations to take into account if you're considering using synthesized lentivirus vectors to stably express a lncRNA in cells. I'll provide in-depth explanations below, but to summarize the bottom line for readers skimming through: the use of lentiviral vectors inherently results in extra sequences and base pairs fused to your lncRNA of interest at the 3' end. This can potentially impact its normal function and mislead results, as you no longer have a fully true representation of the lncRNA, including its subsequent folding and functions. Consider using a transposon-based system for stable cell line generation instead to avoid these issues. If you wish to see data demonstrating how phenotypes can be impacted, Zhang et al. have summarized the message and some results in this publication.
Lentiviral Vector Recap
To begin, let’s go through a high-level recap of how lentiviruses normally operate:
Attachment and entry: The infectious lentivirus particle binds to specific receptors on the target cell surface using its envelope glycoprotein (e.g., gp120 for HIV-1). This interaction triggers the fusion of the viral envelope with the cell membrane, allowing the viral capsid to enter the cell cytoplasm.
Uncoating and reverse transcription: Once inside the cell, the viral capsid undergoes uncoating, releasing the viral RNA genome and enzymes, which include reverse transcriptase. The reverse transcriptase converts the single-stranded RNA genome into a double-stranded DNA (dsDNA) called the provirus.
Nuclear import and integration: The proviral DNA, along with the viral integrase, forms a pre-integration complex (PIC) that is transported into the nucleus. Inside the nucleus, the viral integrase catalyzes the integration of the proviral DNA into the host cell genome.
Viral gene expression and particle assembly: The integrated provirus serves as a template for the production of new viral RNA genomes and translation of viral proteins (Gag, Pol, and Env). These components assemble near the cell membrane to form new viral particles.
Budding and maturation: The immature viral particles bud from the cell membrane, acquiring a lipid envelope containing the viral envelope glycoproteins. After budding, the viral protease cleaves the Gag and Gag-Pol polyproteins, leading to the maturation of the viral particle, which is now capable of infecting new cells.
Lentiviral Vectors for Stable Cell Line Generation
Lentiviral vectors used for creating stable cell lines have been genetically modified to remove the viral genes (gag, pol, and env) from the proviral DNA and replace them with the gene of interest and necessary cis-acting elements (e.g., LTRs, Ψ packaging signal, RRE, cPPT, and WPRE - a table on these below). Due to the removal of the viral genes, the lentiviral vectors result in replication-deficient viral particles - meaning the particles can infect target cells and integrate the gene of interest but cannot produce new, infectious lentiviruses; they are replication-incompetent viruses.
To initially generate useable lentivirus particles given the caveats above, a lentiviral plasmid vector is co-transfected into packaging cells (e.x. HEK293T cells) along with separate plasmids encoding the viral structural and enzymatic proteins (Gag, Pol, and Rev) and the envelope glycoprotein (e.g., VSV-G) needed to make lentivirus. This process provides the necessary components for particle assembly and transduction without the risk of generating replication-competent viruses that contain complete proviral DNA.
Let’s take a look at a schematic for an example lentiviral vector:
The table above outlines some of the basic descriptions of various elements in the vector. Everything between the 5' and 3' LTRs (the viral integrase attachment sites) encodes the proviral DNA, and is also necessary for viral packaging and production.
After co-transfection of the lentiviral vector, packaging plasmid, and envelope plasmid into packaging cells, a lentiviral vector transcript is made starting at the 5'LTR and terminating after the 3'LTR. This transcript participates in various recognition events and complexes (for example, Ψ is recognized by the Gag polyprotein) that mediate its specific packaging into the assembling viral particles. Following a series of steps, the assembled viral particle then buds from the packaging cell's plasma membrane, acquiring the lipid envelope with the embedded envelope glycoprotein. After budding, the immature viral particles undergo maturation through the action of the viral protease. The protease cleaves the Gag and Gag-Pol polyproteins into their constituent proteins, leading to structural rearrangements and the formation of mature, infectious lentiviral particles that are released into the cell culture medium. These lentiviral particles can then be collected, purified, and used to transduce target cells.
Using these purified high-titer lentiviruses for transduction, the viral RNA genome will then be reverse-transcribed and integrated into the host cell genome as proviral DNA. The selected promoter (in the picture above, TRE, a tetracycline-responsive element) drives transcription of the gene of interest's sequence plus all pieces after it (the WPRE element and the selection marker components) down to the poly-A signal near the 3'LTR. For a protein-coding gene, the extra bits on this transcript are inconsequential. Since the start and stop codons will facilitate the correct amino acids being added and terminated within the gene's open reading frame, the correct protein of interest can be generated regardless of the extra base pairs on the transcript.
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However, for non-coding RNA genes, there is no translation taking place. The element of interest is the transcript itself, and it now has the appended sequences for elements like the WPRE and selection markers added to it, which may alter its function. You may be tempted to ask, "Why can't we simply design the vector so there's a poly-A termination signal after the lncRNA sequence, but prior to the WPRE and resistance markers?". If you take into account the information we reviewed above on how lentivirus generation and vectors work, it becomes clear that doing so would prematurely terminate the lentiviral vector RNA, removing the WPRE, selection markers, and 3'LTR. This results in two main issues:
Depending on the vector construct, this can often interfere with lentivirus packaging and production during generation, leading to little-to-no yield.
Even if a high yield of infectious particles with such a transcript packaged was obtained, there would be trouble reverse transcribing and integrating the sequence into the host cell genome after transduction due to the lack of the 3'LTR and reduced stability of the transcript and selection markers.
A Viable Solution: Transposon-based Systems
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So, what's a good solution? A vector that will allow for the proper transcription termination of your lncRNA of interest without losing its ability to integrate into the host genome. In this case, I'll discuss transposons – specifically, transposon plasmids co-expressed with a separate transposase plasmid. Transposons are mobile genetic elements that can move from one location to another within a genome. They consist of a DNA sequence flanked by inverted terminal repeats (ITRs), which are recognized by the transposase enzyme and excised/integrated into the host genome at target sites.
Here is an example of a transposon vector below:
Some examples of commonly used transposons in genetic engineering include the Sleeping Beauty (SB), PiggyBac (PB), and Tol2 transposons. Cells with the integrated sequence can subsequently be selected for using the selection marker (like antibiotic resistance).
Note that a poly-A termination signal (rBG pA in the image above) is possible after the lncRNA sequence, as this won't cause any issues with the transposon or integration into transfected cells. It's also important to mention that not all lncRNAs are necessarily poly-adenylated; some have non-canonical methods and structures that result in transcription termination at the 3' end. However, many of these arise from underlying sequences that can be added to the vector at the correct location.
Summary
In conclusion, when generating stable cell lines to express lncRNAs, it's important to consider the unique challenges posed by their non-coding nature. Lentiviral vectors, while widely used for stable cell line generation, may not be the optimal choice for lncRNAs due to the potential for appending extra sequences to the 3' end of the transcript, which can alter its function and confound experimental results. Transposon-based systems offer a promising alternative, allowing for the proper transcription termination of the lncRNA without the addition of unwanted sequences. By carefully considering these factors and selecting the most appropriate vector system and transcription termination signal/sequence, you can ensure the faithful expression of lncRNAs in stable cell lines and test their potential as valuable tools in cellular processes and developing novel therapeutic strategies.
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