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The lentivirus FLEX conditional Cre-Off gene expression vector combines VectorBuilder’s highly efficient third generation lentiviral vector system with the Cre-responsive FLEX conditional gene expression system to help you achieve permanent integration of Cre-responsive FLEX switch into the host genome. The FLEX Cre-Off switch utilizes two pairs of LoxP-variant recombination sites flanking a gene of interest (GOI) in an arrangement which facilitates robust inactivation of gene expression by Cre-dependent inversion of the GOI.
The FLEX Cre-Off switch consists of two pairs of heterotypic LoxP-variant recombination sites, namely LoxP having the wild type sequence and Lox2272 having a mutated sequence, flanking an ORF. Both LoxP variants are recognized by Cre, but only identical pairs of LoxP sites can recombine with each other and not with any other variant. The LoxP and Lox2272 sites are organized in an alternating fashion, with an antiparallel orientation for each pair. In the absence of Cre recombinase, the ORF can be expressed under the control of the user-selected promoter. In the presence of Cre, the LoxP and Lox2272 sites undergo recombination with the other LoxP and Lox2272 sites respectively, resulting in the inversion of the ORF to an antisense orientation and excision of one from each pair of identical recombination sites. Inversion of the ORF prevents expression of the GOI. Since the ORF is now flanked by two different LoxP-variant sites, no further recombination events will take place even when Cre is present.
The lentiviral vector system is a highly efficient vehicle for introducing genes permanently into mammalian cells. The lentivirus FLEX conditional Cre-Off gene expression vector is first constructed as a plasmid in E. coli with the FLEX Cre-Off switch described above placed in-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 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 into the host genome. The FLEX Cre-Off switch placed in-between the two LTRs during vector construction is permanently inserted into host DNA alongside the rest of viral genome. Gene expression can then be inactivated in the presence of Cre recombinase upon Cre-mediated inversion of the GOI.
By design, lentiviral vectors lack the genes required for viral packaging and transduction (these genes are instead carried by helper plasmids used during virus packaging). As a result, virus produced from lentiviral vectors has 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.
|J Virol. 72:8463 (1998)||The 3rd generation lentivirus vectors|
|Nat Protoc. 1:241 (2006)||Production and purification of lentiviral vectors|
|Gene. 216:55 (1998)||Characterization of LoxP mutants, including Lox2272|
|Nat Biotechnol. 21:562 (2003)||Development of the FLEX switch system|
|J Neurosci. 28:7025 (2008)||Application of a FLEX switch system|
The lentivirus FLEX conditional Cre-Off gene expression vector is designed to achieve Cre-mediated conditional inhibition of gene expression in mammalian cells and animals. Expression of the GOI is initially under the control of the user-selected promoter and can be permanently silenced by co-expression of Cre recombinase, which will invert the GOI to its antisense orientation.
This vector is derived from the third-generation lentiviral vector system. It 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.
Switch-like gene inactivation: Inversion of the user-selected ORF to its antisense orientation in the presence of Cre recombinase prevents any leaky gene expression.
Stable gene inactivation: Treatment with Cre recombinase will permanently invert the user-selected ORF to its antisense orientation. Upon inversion of the ORF to its antisense orientation followed by excision of one from each pair of similar LoxP sites by recombination, the ORF will be flanked by two different LoxP-variant sites which will prevent further recombination events even when Cre is present. This will permanently prevent transcription of the GOI.
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, lentiviral transduction can deliver genes permanently into host cells due to the integration of the viral vector into the host genome.
High viral titer: Our lentiviral vector can be packaged into high titer virus. When lentivirus is obtained through our virus packaging service, titer can reach >109 transducing unit per ml (TU/ml). At this titer, transduction efficiency for cultured mammalian cells can approach 100% when an adequate amount of viral is used.
Very 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 (and even some non-mammalian species) can be transduced. Furthermore, almost any mammalian cell type can be transduced (e.g. dividing cells and non-dividing cells, primary cells and established cell lines, stem cells and differentiated cells, adherent cells and non-adherent cells). Neurons, which are often impervious to conventional transfection, can be readily transduced by our lentiviral vector. Lentiviral vectors packaged with our system have broader tropism than adenoviral vectors (which have low transduction efficiency for some cell types) or MMLV retroviral vectors (which have difficulty transducing non-dividing cells).
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. It is particularly suitable for the generation of transgenic animals with Cre-mediated conditional gene expression.
Safety: The safety of our vector is ensured by two features. One is the partition of genes required for viral packaging and transduction into several helper plasmids; the other is self-inactivation of the promoter activity in the 5' LTR upon vector integration. As a result, it is essentially impossible for replication competent virus to emerge during packaging and transduction. The health risk of working with our vector is therefore minimal.
Limited cargo space: The wildtype lentiviral genome is ~9.2 kb. In our vector, the components necessary for viral packaging and transduction and Cre-mediated recombination occupy ~2.9 kb, which leaves ~6.3 kb to accommodate the user's DNA of interest. When the vector goes beyond this size limit, viral titer can be severely reduced. Our vector is routinely used for inserting several functional elements besides the ORF of the GOI, such as promoter and drug resistance. A large ORF plus these additional elements could exceed 6.3 kb, and the result could be compromised viral production.
Technical complexity: The use of lentiviral 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.
RSV promoter: Rous sarcoma virus promoter. It drives transcription of viral RNA in packaging cells. This RNA is then packaged into live virus.
5' LTR-ΔU3: A deleted version of the HIV-1 5' long terminal repeat. In wildtype lentivirus, 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 in wildtype virus, 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. On our vector, 5' LTR-ΔU3 is deleted for a region that is required for the LTR's promoter activity normally facilitated by the viral transcription factor Tat. This does not affect the production of viral RNA during packaging because the promoter function is supplemented by the RSV promoter engineered upstream of 5'LTR-ΔU3 LTR.
Ψ: HIV-1 packaging signal required for the packaging of viral RNA into virus.
RRE: HIV-1 Rev response element. It allows the nuclear export of viral RNA by the viral Rev protein during viral packaging.
cPPT: HIV-1 Central polypurine tract. It creates a "DNA flap" that increases nuclear import of the viral genome during target cell infection. This improves vector integration into the host genome, resulting in higher transduction efficiency.
Promoter: The promoter driving your GOI is placed here.
Lox2272: Recombination site for Cre recombinase. Mutated Lox site with two base substitutions of wild type LoxP. Incompatible with LoxP sites. When Cre is present, the LoxP and LoxP2272 sites will be cut and recombine with compatible sites.
LoxP: Recombination site for Cre recombinase. Incompatible with Lox2272 sites. When Cre is present, the LoxP and Lox2272 sites will be cut and recombine with compatible sites.
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 GOI is placed here, in a sense orientation.
WPRE: Woodchuck hepatitis virus posttranscriptional regulatory element. It enhances transcriptional termination in the 3' LTR during viral RNA transcription, which leads to higher levels of functional viral RNA in packaging cells and hence greater viral titer. It also enhances transcriptional termination during the transcription of the user's GOI on the vector, leading to their higher expression levels.
mPGK promoter: Mouse phosphoglycerate kinase 1 gene promoter. It drives the ubiquitous expression the downstream marker gene.
Marker: A drug selection gene (such as neomycin resistance), a visually detectable gene (such as EGFP), or a dual-reporter gene (such as EGFP/Neo). This allows cells transduced with the vector to be selected and/or visualized.
3' LTR-ΔU3: A truncated version of the HIV-1 3' long terminal repeat that deletes the U3 region. This leads to the self-inactivation of the promoter activity of the 5' LTR upon viral vector integration into the host genome (since the 3' LTR is copied onto 5' LTR during viral integration). The polyadenylation signal contained in 3' LTR-ΔU3 serves to terminates all upstream transcripts produced both during viral packaging and after viral integration into the host genome.
SV40 early pA: Simian virus 40 early polyadenylation signal. It further facilitates transcriptional termination after the 3' LTR during viral RNA transcription during packaging. This elevates the level of functional viral RNA in packaging cells, thus improving viral titer.
Ampicillin: Ampicillin resistance gene. It allows the plasmid to be maintained by ampicillin selection in E. coli.
pUC ori: pUC origin of replication. Plasmids carrying this origin exist in high copy numbers in E. coli.