MSCV Retrovirus Non-Coding RNA Expression Vector
The MSCV (Murine stem cell virus) retroviral vector system is a highly efficient viral vehicle for permanently introducing non-coding RNAs of interest in embryonic stem (ES) cells, embryonal carcinoma (EC) cells and hematopoietic stem (HS) cells along with several other mammalian cell lines. Non-coding RNAs include a wide variety of short (<30 nucleotides) and long (>200 nucleotides) functional RNA molecules such as microRNAs (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 MSCV retrovirus non-coding RNA expression vector uses the ubiquitous promoter function in the 5’ LTR (long terminal repeat) of the MSCV retroviral genome to drive the expression of the user-selected non-coding RNA gene, which is mediated by RNA polymerase ll-dependent transcription. For RNA polymerase ll-mediated transcription, the start site is typically in the 3’ region of the promoter while the termination site is within the 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.
An MSCV retroviral vector is first constructed as a plasmid in E. coli. The non-coding RNA of interest is cloned between the two LTRs during vector construction. It is then transfected into packaging cells along with several helper plasmids. Inside the packaging cells, vector DNA located between the two 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 in the host genome. Any gene(s) that were placed in-between the two LTRs during vector cloning are permanently inserted into the host DNA alongside the rest of the viral genome.
By design, MSCV retroviral vectors lack the genes required for viral packaging and transduction (these genes are carried by helper plasmids or integrated into packaging cells instead). As a result, viruses produced from the vectors have 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|
|PLoS One. 8:e77070 (2013)||Retrovirus-mediated expression of long non-coding RNA|
|Exp Hematol. 31: 1007 (2003)||Review on retrovirus-mediated gene expression|
|Gene Ther. 1: 136 (1994)||Development of the MSCV vector series|
Our vector 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 including ES, EC and HS 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, retroviral transduction can deliver genes permanently into host cells due to integration of the viral vector into the host genome.
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 commonly used mammalian species such as human, mouse and rat can be transduced. Additionally, the presence of a strategically designed 5’LTR derived from the PCMV virus in the MSCV retroviral vector helps to achieve high level expression of target genes in ES, EC and HS cells. This offers a distinct advantage over MMLV retroviral vectors which show a restricted expression of target genes in ES and EC cells. However, our MSCV vector has difficulty transducing non-dividing cells (see disadvantages below).
Large cargo space: The genome for the MSCV retroviral vector is ~8 kb. In our vector, the components necessary for viral packaging and transduction occupy ~1.9 kb, which leaves ~6.1 kb to accommodate the user's DNA of interest. Because our vector is designed for the insertion of only a non-coding RNA sequence, this cargo space is sufficient for most applications.
High-level expression: The 5' LTR contains a strong ubiquitous promoter that drives high-level expression of the user's non-coding RNA 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 partitioning genes required for viral packaging and transduction into several helper plasmids or integrating them into packaging cells. As a result, live virus produced from our vector is replication incompetent.
Dependence on 5' LTR promoter: Expression of the non-coding RNA in our vector is driven by the ubiquitous promoter function in the 5' LTR. This is a distinct disadvantage as compared to our lentiviral vectors which allow the user to put in their own promoter to drive their non-coding RNA of interest.
Moderate viral titer: Viral titer from our vector reaches ~107 TU/ml in the supernatant of packaging cells without further concentration. This is about an order of magnitude lower than our lentiviral vectors.
Difficulty transducing non-dividing cells: MSCV has difficulty transducing non-dividing cells.
Technical complexity: The use of MSCV retroviral 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.
MSCV 5’ LTR: 5' long terminal repeat from PCC4-cell-passaged myeloproliferative sarcoma virus (PCMV). The LTRs carry both promoter and polyadenylation function, such that 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. The 5’ LTR derived from the PCMV retrovirus in the MSCV vector has been strategically modified to drive the transcriptional activation of target genes in pluripotent cell lines such as ES or EC cells, unlike MMLV retroviral vectors.
MSCV Ψ+： Murine embryonic stem cell virus packaging signal required for packaging of viral RNA into virus.
Non-coding RNA: Your non-coding RNA of interest is placed here. Its expression is driven by ubiquitous promoter function in the 5’ LTR.
MSCV 3’ LTR: 3' long terminal repeat from PCMV. It allows packaging of viral RNA into virus and facilitates transcription termination and mRNA polyadenylation in ES cells and other cell types.
pUC ori: pUC origin of replication. Plasmids carrying this origin exist in high copy numbers in E. coli.
Ampicillin: Ampicillin resistance gene. It allows the plasmid to be maintained by ampicillin selection in E. coli.