gRNA and Cas9 Coexpression
CRISPR-Based Gene Activation
MMLV Retrovirus Gene Expression Vector
The MMLV retroviral vector system is an efficient vehicle for introducing genes permanently into mammalian cells. It became particularly popular as a gene delivery method for making iPS cells.
MMLV retroviral vectors are derived from Moloney murine leukemia virus, which is a member of the retrovirus family. Wildtype MMLV virus has a plus-strand linear RNA genome.
An MMLV retroviral vector is first constructed as a plasmid in E. coli. It is then transfected into packaging cells along with several helper plasmids. Inside the packaging cells, vector DNA located between the two long terminal repeats (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 construction are 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.
|Exp Hematol. 31:1007 (2003)||Review|
|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|
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, 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, from commonly used mammalian species such as human, mouse and rat can be transduced. Furthermore, many 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 an ORF, 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 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 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 gene 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 ~107/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 technical demanding and time consuming relative to conventional plasmid transfection.
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.
Kozak: Kozak consensus sequence. It is placed in front of the start codon of the ORF of interest because it is believed to facilitate translation initiation in eukaryotes.
ORF: The open reading frame of your gene of 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 terminates the transcript from the upstream ORF.
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.