MMLV Retrovirus Non-Coding RNA Expression Vector
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.