MMLV Retrovirus Non-Coding RNA Expression Vector

Overview

The MMLV retrovirus 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 MMLV retrovirus non-coding RNA expression vector uses the ubiquitous promoter function in the 5' LTR of the MMLV retroviral genome to drive the expression of the user-selected non-coding RNA gene, which is mediated by RNA polymerase II-dependent transcription. 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 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 MMLV retroviral vector is first constructed as a plasmid in E. coli. The non-coding RNA of interest is cloned between the two long terminal repeats (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 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. The non-coding RNA sequence that was placed in-between the two LTRs during vector construction is permanently inserted into host DNA alongside the rest of viral genome.

By design, MMLV 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.

References Topic
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
J Virol. 61:1639 (1987) Extended packaging signal increases the titer of MMLV vectors
Gene Ther. 7:1063 (2000) Tropism of MMLV vectors depends on packaging cell lines
Nat Protoc. 6:346 (2011) Tropism of MMLV vectors depends on packaging plasmids

Highlights

The MMLV retrovirus non-coding RNA expression 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, efficient vector integration into the host genome, and high-level transgene expression.

Advantages

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 all commonly used mammalian species such as human, mouse and rat can be transduced. Furthermore, almost any mammalian cell types can be transduced, though our vector has difficulty transducing non-dividing cells (see disadvantages below).

Large cargo space: The wildtype MMLV retroviral genome is ~8 kb. In our vector, the components necessary for viral packaging and transduction occupy ~2.5 kb, which leaves ~5.5 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.

Disadvantages

Dependence on 5' LTR promoter: Expression of the non-coding RNA of interest 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 gene of interest.

Moderate viral titer: Viral titer from our vector reach ~108 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: Our vector has difficulty transducing non-dividing cells.

Technical complexity: The use of MMLV 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.

Key components

5' MoMuLV LTR: MMLV retrovirus 5' long terminal repeat. In wildtype MMLV retrovirus, 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 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.

ψ plus pack2: MMLV retrovirus packaging signal required for the packaging of viral RNA into virus.

Non-coding RNA: The non-coding RNA of your interest is placed here. Its expression is driven by the ubiquitous promoter function in the 5' LTR.

3' MoMuLV LTR: MMLV retrovirus 3' long terminal repeat. The polyadenylation signal contained in 3' LTR serves to terminate the transcript from the upstream non-coding RNA.

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.

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