Retroviral vectors for constitutive gene expression

A system composed of three retroviral cloning vectors, as well as 5' and 3' LNCX sequencing/PCR primers. The system is suitable for use with the Retro-X system (Cat No. 631508). The pLNCX2, pLPCX, and pLHCX retroviral vectors contain a gene encoding resistance to neomycin, puromycin, or hygromycin, respectively. With these vectors, a gene of interest is expressed under control of the CMV promoter. The vectors are excellent tools for expression studies in primary cell lines, explants, or whole animals.

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Schematic of LRCX retroviral vectors

Schematic of LRCX retroviral vectors.

This product is a plasmid vector for producing a high expression efficiency retrovirus vector. By transfecting this vector into an appropriate packaging cell line, the vector transiently or stably expresses transcribed products containing the virus-packaging signal (Ψ) and target gene. This vector possesses LTR and Ψ (viral packaging signal), but not the structural genes necessary for particle formation and replication (sequences gag, pol, and env). Because this vector contains an intron with high splicing capacity, it yields high transcription efficiency. This vector contains the HCMV IE promoter (which can produce high-titer retroviruses) within the U3 region of the 5' LTR, and a neomycin resistant gene as a selective marker.

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Vector map for pDON-5

Vector map for pDON-5.
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This product is a plasmid vector for producing a high expression efficiency retrovirus vector. By transfecting this vector into an appropriate packaging cell line, the vector transiently or stably expresses transcribed products containing the virus-packaging signal (Ψ), target gene and selective marker. This vector possesses LTR and Ψ (viral packaging signal), but not the structural genes necessary for particle formation and replication (sequences gag, pol, and env). Because this vector contains an intron with high splicing capacity, it yields high transcription efficiency. This vector contains the HCMV IE promoter (which can produce high-titer retroviruses) within the U3 region of the 5' LTR, and a neomycin resistant gene as a selective marker.

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Vector map for pDON-5 Neo

Vector map for pDON-5 Neo.

pDON-AI-2 DNA enables production of a retrovirus vector with high transfer efficiency. By transfecting the vector into an appropriate packaging cell line, the vector expresses transient or stable transcribed product containing virus-packaging signal (Ψ) and target gene. This vector possesses LTR and Ψ (viral packaging signal), but not the structural gene necessary for particle formation and replication, which are gag, pol, and env, and contains HCMV IE promoter within the U3 region of 5'LTR. The production of 2–8-fold higher-titer virus can be achieved by transfecting a version of pDON-AI-2 DNA containing a target gene compared using conventional retrovirus vector.

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Vector map for pDON-AI-2

Vector map for pDON-AI-2.
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pDON-AI-2 DNA enables production of a retrovirus vector with high transfer efficiency. By transfecting the vector into an appropriate packaging cell line, the vector expresses transient or stable transcribed product containing virus-packaging signal (Ψ) and target gene. This vector possesses LTR and Ψ (viral packaging signal), but not the structural gene necessary for particle formation and replication, which are gag, pol, and env, and contains HCMV IE promoter within the U3 region of 5'LTR. pDON-AI-2 Neo DNA contains a neomycin resistant gene as a selective marker. The production of 2–8-fold higher-titer virus can be achieved by transfecting a version of pDON-AI-2 DNA containing a target gene compared using conventional retrovirus vector.

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Vector map for pDON-AI-2-Neo

Vector map for pDON-AI-2-Neo.
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pLNCX2 is designed for retroviral gene delivery and expression. Upon transfection into a packaging cell line, pLNCX2 can transiently express, or integrate and stably express, a transcript containing the Ψ viral packaging signal, a selectable marker, and the geneof interest, which is cloned into the MCS under the control of the immediate early promoter of human cytomegalovirus (P_CMV). The 5' viral LTR drives expression of the neomycin resistance gene for antibiotic selection in eukaryotic cells.

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Schematic of LRCX retroviral vectors

Schematic of LRCX retroviral vectors.

pLXIN is a bicistronic retroviral vector containing an internal ribosome entry site (IRES). The 5' LTR thus regulates expression of a gene of interest and the neomycin resistance gene on the same bicistronic message.

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Schematic of LRCX retroviral vectors

Schematic of LRCX retroviral vectors.

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pLXIN Vector map

pLXIN Vector map.

pLXSN is designed for retroviral gene delivery and expression of gene of interest. It contains the retroviral packaging sequence (Ψ+) and so, when transfected into a cell line expressing the retroviral structural proteins (gag, pol, and env), leads to packaging of viral particles. The 5' viral LTR in this vector contains a viral promoter that controls expression of the gene of interest.

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Retroviral vectors available from Clontech

Retroviral vectors available from Takara Bio. Not all vectors are shown. Some retroviral vectors are not sold separately.

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Schematic of LRCX retroviral vectors

Schematic of LRCX retroviral vectors.

This product is a plasmid vector for producing a high expression efficiency retrovirus vector. By transfecting this vector into an appropriate packaging cell line, the vector transiently or stably expresses transcribed products containing the virus-packaging signal (Ψ), target gene and selective marker. This vector possesses LTR and Ψ (viral packaging signal), but not the structural genes necessary for particle formation and replication (sequences gag, pol, and env). Because this vector contains an intron with high splicing capacity, it yields high transcription efficiency. Gene expression two to eight-fold higher than pDON-Al-2 series can be expected.

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Vector map for pMEI-5

Vector map for pMEI-5.
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This product is a plasmid vector for producing a high expression efficiency retrovirus vector. By transfecting this vector into an appropriate packaging cell line, the vector transiently or stably expresses transcribed products containing the virus-packaging signal (Ψ), target gene and selective marker. This vector possesses LTR and Ψ (viral packaging signal), but not the structural genes necessary for particle formation and replication (sequences gag, pol, and env). Because this vector contains an intron with high splicing capacity, it yields high transcription efficiency. pMEI-5 Neo DNA (Cat.# 3655) contains the neomycin resistance gene as a selective marker. Gene expression two to eight-fold higher than pDON-Al-2 series can be expected.

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Vector map for pMEI-5 Neo

Vector map for pMEI-5 Neo.
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The pQCXIN Retroviral Vector is a self-inactivating retroviral Q expression vector that is optimized to eliminate promoter interference from the upstream LTR in the integrated provirus. It expresses a target gene along with the gene for the neomycin resistance marker from a single, bicistronic message. The CMV promoter present in the 5' LTR produces high viral titers in standard packaging cell lines. A deletion in the 3' LTR provides the self-inactivating feature after reverse transcription of the expression cassette. This vector is useful for applications in which high titers and high-level gene expression are desired.

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Composite retroviral vector map of Retro-X Q Vector Set Set

Composite retroviral vector map of Retro-X Q Vector Set Set In this generalized version of the Q Vector Map, the common elements of all the vectors are represented. Immediately downstream of the CMV immediate early promoter, the multiple cloning site (MCS) is followed by a eukaryotic IRES that ensures a second ORF (an antibiotic resistance marker or another gene in the case of pQCXIX) is cotranscribed with the gene cloned into the MCS. The expression cassette has all of the essential elements for retroviral integration and expression.

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Self-inactivation mechanism of the Retro-X Q retroviral vectors

Self-inactivation mechanism of the Retro-X Q retroviral vectors. The plus strand retroviral RNA (blue) is reverse transcribed. During integration, a circular intermediate is formed that results in duplication of the deletion in the U3 region of the 3' LTR. This inactivates the CMV/MSV hybrid promoter in the 5' LTR of the retrovirus so that transcription can only be driven from the internal promoter, P_{CMV IE}.

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Retroviral vectors available from Clontech

Retroviral vectors available from Takara Bio. Not all vectors are shown. Some retroviral vectors are not sold separately.

The pQCXIX Retroviral Vector is a self-inactivating retroviral expression vector that is optimized to eliminate promoter interference from the upstream LTR in the integrated provirus. It expresses two target genes from a single, bicistronic message. The CMV promoter present in the 5' LTR produces high viral titers in standard packaging cell lines. A deletion in the 3' LTR provides the self-inactivating feature after reverse transcription of the expression cassette. This vector is useful for applications in which high titers and high-level bicistronic gene expression are desired.

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Composite retroviral vector map of Retro-X Q Vector Set Set

Composite retroviral vector map of Retro-X Q Vector Set Set In this generalized version of the Q Vector Map, the common elements of all the vectors are represented. Immediately downstream of the CMV immediate early promoter, the multiple cloning site (MCS) is followed by a eukaryotic IRES that ensures a second ORF (an antibiotic resistance marker or another gene in the case of pQCXIX) is cotranscribed with the gene cloned into the MCS. The expression cassette has all of the essential elements for retroviral integration and expression.

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Self-inactivation mechanism of the Retro-X Q retroviral vectors

Self-inactivation mechanism of the Retro-X Q retroviral vectors. The plus strand retroviral RNA (blue) is reverse transcribed. During integration, a circular intermediate is formed that results in duplication of the deletion in the U3 region of the 3' LTR. This inactivates the CMV/MSV hybrid promoter in the 5' LTR of the retrovirus so that transcription can only be driven from the internal promoter, P_{CMV IE}.

pRetro-Lib, derived from Moloney murine leukemia virus (MoMuLV), is designed for delivery and expression of retroviral libraries or genes. Upon transfection into a packaging cell line, pRetro-Lib can transiently express, or integrate and stably express, a transcript containing (psi, the extended viral packaging signal) and the gene of interest. The 5' viral LTR in this vector contains a viral promoter that controls expression of genes cloned into the MCS.

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pRetro-Lib Vector map

pRetro-Lib Vector map. A retroviral expression vector for cloning libraries. Suitable for use with the SMART cDNA Library Construction Kit (Cat. # 634901).

Required Products

Cat. #	Product	Size	Price	License	Quantity	Details
639206	Advantage® 2 PCR Kit	100 Rxns	$445.00
This kit is based on Takara's Advantage 2 Polymerase Mix. It contains all reagents needed for a hot-start PCR, including control template and primer mix. This kit is ideal for PCR applications that require high fidelity, such as cDNA amplification or library construction. Enough reagents are supplied for 100 PCR reactions of 50 μl each. Documents Components Image Data Back Amplification of a fragment of the rare tumor necrosis factor receptor II (TNFR II) cDNA with Advantage 2 Polymerase Mix and a competitor's Taq polymerase mix Amplification of a fragment of the rare tumor necrosis factor receptor II (TNFR II) cDNA with Advantage 2 Polymerase Mix and a competitor's Taq polymerase mix. 5 μl of PCR products were run on a 1.1% agarose/EtBr gel. Lane 1: The 0.4-kb TNFR II fragment is readily obtained with Advantage 2. Lane 2: No product is seen with Taq polymerase. Lane M: DNA size marker Back Amplification of various large templates using Advantage 2 Polymerase Mix Amplification of various large templates using Advantage 2 Polymerase Mix. 1–3 μl of each PCR product was run on a 1.1% agarose/EtBr gel. Lane 1: 2.5-kb E. coli DNA polymerase gene amplified from genomic DNA. Lane 2: 3.5-kb bovine pancreatic trypsin inhibitor gene amplified from calf thymus genomic DNA. Lane 3: 5.9-kb human IL-1β gene amplified from human genomic DNA. Lane 4: 8.5-kb human titin cDNA amplified from a SMART Human Skeletal Muscle cDNA library. Lane 5: 18.5-kb λ insert amplified from a λ clone. Lane M: λ/Hind III DNA size marker. Back Advantage 2 PCR KitCatalog No Advantage 2 PCR Kit Catalog No.639206.

The Retro-X Q Vector set provides four retroviral expression vectors that are optimized to eliminate promoter interference from the upstream LTR in the integrated provirus. Each vector expresses a target gene along with a different selection marker from a single, bicistronic message. The CMV promoter present in the 5' LTR produces high viral titers in standard packaging cell lines. A LacZ Control Vector pQCLIN is included in the set. Also included are Retro-X Q Primers that encode sequences flanking the multiple cloning site.

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Composite retroviral vector map of Retro-X Q Vector Set Set

Composite retroviral vector map of Retro-X Q Vector Set Set In this generalized version of the Q Vector Map, the common elements of all the vectors are represented. Immediately downstream of the CMV immediate early promoter, the multiple cloning site (MCS) is followed by a eukaryotic IRES that ensures a second ORF (an antibiotic resistance marker or another gene in the case of pQCXIX) is cotranscribed with the gene cloned into the MCS. The expression cassette has all of the essential elements for retroviral integration and expression.

Back

Self-inactivation mechanism of the Retro-X Q retroviral vectors

Self-inactivation mechanism of the Retro-X Q retroviral vectors. The plus strand retroviral RNA (blue) is reverse transcribed. During integration, a circular intermediate is formed that results in duplication of the deletion in the U3 region of the 3' LTR. This inactivates the CMV/MSV hybrid promoter in the 5' LTR of the retrovirus so that transcription can only be driven from the internal promoter, P_{CMV IE}.