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The MSCV (Murine stem cell virus) retroviral vector system is a highly efficient viral vehicle for achieving robust expression of genes of interest in embryonic stem (ES) cells, embryonal carcinoma (EC) cells and hematopoietic stem (HS) cells along with several other mammalian cell lines.
MSCV retroviral vectors are derived from the murine PCC4-cell passaged myeloproliferative sarcoma virus (PCMV) based MESV retroviral vectors and Moloney murine leukemia virus (MMLV) based LN retroviral vectors. The inclusion of an extended hybrid packaging signal derived from the LN vectors helps to achieve higher viral titer with the MSCV retroviral vector compared to traditional retroviral vectors. Additionally, the presence of a strategically designed 5’LTR derived from a modified PCMV virus in the MSCV retroviral vector contributes to transcriptional activation of target genes in pluripotent cell lines such as ES or EC cells. This helps to overcome the restricted expression of target genes in ES and EC cells, typically observed with MMLV retroviral vectors.
An MSCV 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 cloning are permanently inserted into host DNA alongside the rest of 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.
|Gene Ther. 1:136 (1994)||Development of the MSCV vector series|
|Proc Natl Acad Sci U S A. 87:9202 (1990)||Development of the MESV retroviral vector|
|Biotechniques 7:980 (1989)||Development of LNL6 retroviral vectors|
|Proc Natl Acad Sci U S A. 83:3292 (1986)||Characterization of retroviral mutants expressed in EC cells|
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 cargo limit for the MSCV retroviral vector is ~8.3 kb. In our vector, the components necessary for viral packaging and transduction occupy ~2-2.5 kb, which leaves ~5.8-6.3 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 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 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 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.
MSCV 3' LTR: 3' long terminal repeat from PCC4-cell-passaged myeloproliferative sarcoma virus (PCMV). Allows packaging of viral RNA into virus. Also 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.