Lentivirus Non-Coding RNA Expression Vector
The lentivirus non-coding RNA expression vector is a highly efficient vehicle for permanently introducing non-coding RNAs of interest in mammalian cells. Non-coding RNAs include a wide variety of short (<30 nucleotides) and long (>200 nucleotides) functional RNA molecules such as micro RNAs (miRNAs), small interfering RNAs (siRNAs), piwi-interacting RNAs (piRNAs), small nuclear RNAs (snRNAs), small nucleolar RNAs (snoRNAs), large intergenic non-coding RNAs (lincRNAs), intronic long non-coding RNAs (intronic lncRNAs), natural antisense transcripts (NATs), enhancer RNAs (eRNAs) and promoter-associated RNAs (PARs), none of which are translated into proteins, however have been found to play important roles in many cellular processes such as DNA replication, epigenetic regulation, transcriptional and post-transcriptional regulation and translation regulation.
The lentivirus non-coding RNA expression vector uses an RNA polymerase II promoter to drive the expression of the user-selected non-coding RNA gene. This allows the use of tissue-specific, inducible, or variable-strength promoters, enabling a variety of experimental applications. For RNA polymerase II-mediated transcription, the start site is typically in the 3' region of the promoter while the termination site is within the downstream polyA signal sequence. As a result, the transcript generated from this vector does not correspond precisely to the selected non-coding RNA gene, but contains some additional sequences both upstream and downstream. For the lentiviral vector, internal polyadenylation signal is not suggested to be placed between the LTRs for each individual expression cassette, as this would inhibit virus packaging. Therefore, sequence downstream of the non-coding RNA till 3' LTR will be transcribed along with the non-coding RNA which may affect the non-coding RNA function.
The lentivirus non-coding RNA expression vector is first constructed as a plasmid in E. coli. The non-coding RNA of interest along with a user-selected promoter is cloned between the two long terminal repeats (LTRs) during vector construction. The vector is then transfected into packaging cells along with several helper plasmids. Inside the packaging cells, vector DNA located between the LTRs is transcribed into RNA, and viral proteins expressed by the helper plasmids further package the RNA into virus. Live virus is then released into the supernatant, which can be used to infect target cells directly or after concentration.
When the virus is added to target cells, the RNA cargo is shuttled into cells where it is reverse transcribed into DNA and randomly integrated into the host genome. The non-coding RNA sequence placed in-between the two LTRs during vector construction is permanently inserted into host DNA alongside the rest of viral genome.
By design, lentiviral vectors lack the genes required for viral packaging and transduction (these genes are instead carried by helper plasmids used during virus packaging). As a result, virus produced from lentiviral vectors has the important safety feature of being replication incompetent (meaning that they can transduce target cells but cannot replicate in them).
For further information about this vector system, please refer to the papers below.
|Cell. 157:77 (2014)||Review on non-coding RNAs|
|Front Genet. 6:2 (2015)||Review on functionality of non-coding RNAs|
|Int J Oncol. 48:1509 (2016)||Lentivirus-mediated expression of long non-coding RNA|
|J Virol. 72:8463 (1998)||The 3rd generation lentivirus vectors|
|J Virol. 72:9873 (1998)||Self-inactivating lentivirus vectors|
|Nat Protoc. 1:241 (2006)||Production and purification of lentiviral vectors|
The lentivirus non-coding RNA expression vector is derived from the third-generation lentiviral vector system. It is optimized for high copy number replication in E. coli, high-titer packaging of live virus, efficient viral transduction of a wide range of cells, efficient vector integration into the host genome, and high-level transgene expression.
Permanent integration of vector DNA: Conventional transfection results in almost entirely transient delivery of DNA into host cells due to the loss of DNA over time. This problem is especially prominent in rapidly dividing cells. In contrast, lentiviral transduction can deliver non-coding RNAs permanently into host cells due to the integration of the viral vector into the host genome.
High viral titer: Our lentiviral vector can be packaged into high titer virus. When lentivirus is obtained through our virus packaging service, titer can reach >109 transducing unit per ml (TU/ml). At this titer, transduction efficiency for cultured mammalian cells can approach 100% when an adequate amount of viral is used.
Very broad tropism: Our packaging system adds the VSV-G envelop protein to the viral surface. This protein has broad tropism. As a result, cells from all commonly used mammalian species (and even some non-mammalian species) can be transduced. Furthermore, almost any mammalian cell type can be transduced (e.g. dividing cells and non-dividing cells, primary cells and established cell lines, stem cells and differentiated cells, adherent cells and non-adherent cells). Neurons, which are often impervious to conventional transfection, can be readily transduced by our lentiviral vector. Lentiviral vectors packaged with our system have broader tropism than adenoviral vectors (which have low transduction efficiency for some cell types) or MMLV retroviral vectors (which have difficulty transducing non-dividing cells).
Customizable internal promoter: Our vector is designed to self-inactivate the promoter activity in its 5' LTR upon integration into the genome. As a result, users can put in their own promoter to drive their non-coding RNA of interest within the vector. This is a distinct advantage over our MMLV retrovirus vectors, which rely on the promoter function of 5' LTR to drive the ubiquitous expression of the gene of interest.
Relative uniformity of gene delivery: Generally, viral transduction can deliver vectors into cells in a relatively uniform manner. In contrast, conventional transfection of plasmid vectors can be highly non-uniform, with some cells receiving a lot of copies while other cells receiving few copies or none.
Effectiveness in vitro and in vivo: While our vector is mostly used for in vitro transduction of cultured cells, it can also be used to transduce cells in live animals.
Safety: The safety of our vector is ensured by two features. One is the partition of genes required for viral packaging and transduction into several helper plasmids; the other is self-inactivation of the promoter activity in the 5' LTR upon vector integration. As a result, it is essentially impossible for replication competent virus to emerge during packaging and transduction. The health risk of working with our vector is therefore minimal.
Limited cargo space: The wildtype lentiviral genome is ~9.2 kb. In our vector, the components necessary for viral packaging and transduction occupy ~2.8 kb, which leaves ~6.4 kb to accommodate the user's DNA of interest. When the vector goes beyond this size limit, viral titer can be severely reduced. Our vector is routinely used for inserting several functional elements besides the non-coding RNA of interest, such as promoter and drug resistance. A large non-coding RNA plus these additional elements could exceed 6.4 kb, and the result could be compromised viral production.
Technical complexity: The use of lentiviral vectors requires the production of live virus in packaging cells followed by the measurement of viral titer. These procedures are technically demanding and time consuming relative to conventional plasmid transfection.
RSV promoter: Rous sarcoma virus promoter. It drives transcription of viral RNA in packaging cells. This RNA is then packaged into live virus.
5' LTR-ΔU3: A deleted version of the HIV-1 5' long terminal repeat. In wildtype lentivirus, 5' LTR and 3' LTR are essentially identical in sequence. They reside on two ends of the viral genome and point in the same direction. Upon viral integration, the 3' LTR sequence is copied onto the 5' LTR. The LTRs carry both promoter and polyadenylation function, such that in wildtype virus, the 5' LTR acts as a promoter to drive the transcription of the viral genome, while the 3' LTR acts as a polyadenylation signal to terminate the upstream transcript. On our vector, Δ5' LTR is deleted for a region that is required for the LTR's promoter activity normally facilitated by the viral transcription factor Tat. This does not affect the production of viral RNA during packaging because the promoter function is supplemented by the RSV promoter engineered upstream of Δ5' LTR.
Ψ: HIV-1 packaging signal required for the packaging of viral RNA into virus.
RRE: HIV-1 Rev response element. It allows the nuclear export of viral RNA by the viral Rev protein during viral packaging.
cPPT: HIV-1 Central polypurine tract. It creates a "DNA flap" that increases nuclear importation of the viral genome during target cell infection. This improves vector integration into the host genome, resulting in higher transduction efficiency.
Promoter: The promoter driving your non-coding RNA of interest is placed here.
Non-coding RNA: The non-coding RNA of your interest is placed here.
WPRE: Woodchuck hepatitis virus posttranscriptional regulatory element. It enhances viral RNA stability in packaging cells, leading to higher titer of packaged virus.
mPGK promoter: Mouse phosphoglycerate kinase 1 gene promoter. It drives the ubiquitous expression the downstream marker gene.
Marker: A drug selection gene (such as neomycin resistance), a visually detectable gene (such as EGFP), or a dual-reporter gene (such as EGFP/Neo). This allows cells transduced with the vector to be selected and/or visualized.
3' LTR-ΔU3: A truncated version of the HIV-1 3' long terminal repeat that deletes the U3 region. This leads to the self-inactivation of the promoter activity of the 5' LTR upon viral vector integration into the host genome (due to the fact that 3' LTR is copied onto 5' LTR during viral integration). The polyadenylation signal contained in ΔU3/3' LTR serves to terminates all upstream transcripts produced both during viral packaging and after viral integration into the host genome.
SV40 early pA: Simian virus 40 early polyadenylation signal. It further facilitates transcriptional termination after the 3' LTR during viral RNA transcription during packaging. This elevates the level of functional viral RNA in packaging cells, thus improving viral titer.
Ampicillin: Ampicillin resistance gene. It allows the plasmid to be maintained by ampicillin selection in E. coli.
pUC ori: pUC origin of replication. Plasmids carrying this origin exist in high copy numbers in E. coli.