pTRE-Cycle2 is a bidirectional, Tet-inducible expression vector that allows you to reversibly regulate (i.e., cycle) the amount of a protein of interest in mammalian cells while coexpressing the red fluorescent protein mCherry. Expression of both proteins is tightly regulated by the bidirectional, TRE-based promoter, P_Tight-Bi. Expression of the gene of interest is controlled by an additional mechanism, since the resulting protein is fused to an N-terminal ProteoTuner destabilization domain (DD), which causes rapid proteasomal degradation of any protein to which it is fused. The degradation process can be reversed repeatedly by adding the stabilizing ligand Shield1 and then removing it from the culture medium. mCherry is coexpressed with the tagged protein, but does not contain a DD-tag, and thus is subject only to Tet-based regulation. The vector is intended for use with any Tet-On or Tet-Off Advanced Expression System. To select stable cell lines, the vector must be cotransfected with one of the provided linear selection markers.

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Inducible expression of two proteins of interest, one with a DD tag and one induced by tetracycline

Inducible expression of two proteins of interest, one with a DD tag and one induced by tetracycline. pTRE-Cycle2 and pTRE-Cycle3 allow you to inducibly coexpress a red or green fluorescent protein along with your DD-tagged protein of interest.

pTRE-Cycle3 is a bidirectional, Tet-inducible expression vector that allows you to reversibly regulate (i.e., cycle) the amount of a protein of interest in mammalian cells while coexpressing the fluorescent protein ZsGreen1. Expression of both proteins is tightly regulated by the bidirectional, TRE-based promoter, P_Tight-Bi.Expression of the gene of interest is controlled by an additional mechanism, since the resulting protein is fused to an N-terminal ProteoTuner destabilization domain (DD), which causes rapid proteasomal degradation of any protein to which it is fused. The degradation process can be reversed repeatedly by adding the stabilizing ligand Shield1 and then removing it from the culture medium. ZsGreen1 is coexpressed with the tagged protein, but does not contain a DD-tag, and thus is subject only to Tet-based regulation. The vector is intended for use with any Tet-On or Tet-Off Advanced Expression System. To select stable cell lines, the vector must be cotransfected with one of the provided linear selection markers.

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Independent inducible expression of two proteins in HEK 293 cells is shown for red and green fluorescent proteins and also for Sox2 and Oct4

Independent inducible expression of two proteins in HEK 293 cells is shown for red and green fluorescent proteins (Panel A) and also for Sox2 and Oct4 (Panel B).

pTRE-Cycle1 is a bidirectional, Tet-inducible expression vector that allows you to reversibly regulate (i.e., cycle) the amount of a protein of interest in mammalian cells while coexpressing a second protein that is not cycled. Expression of both proteins is tightly regulated by the bidirectional, TRE-based promoter, P_Tight-Bi.Expression of the first gene of interest is controlled by an additional mechanism, since the resulting protein is fused to an N-terminal ProteoTuner^TM destabilization domain (DD), which causes rapid proteasomal degradation of any protein to which it is fused. The degradation process can be reversed repeatedly by adding the stabilizing ligand Shield1 and then removing it from the culture medium. The second gene of interest, which is cloned into a separate multiple cloning site, will be coexpressed with the tagged protein, but will not acquire a DD-tag, and thus is subject only to Tet-based regulation. The vector is intended for use with any Tet-On or Tet-Off Advanced Expression System. To select stable cell lines, the vector must be cotransfected with one of the provided linear selection markers.

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Inducible expression of two proteins of interest, one with a DD tag and one induced by tetracycline

Inducible expression of two proteins of interest, one with a DD tag and one induced by tetracycline. pTRE-Cycle2 and pTRE-Cycle3 allow you to inducibly coexpress a red or green fluorescent protein along with your DD-tagged protein of interest.

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Independent inducible expression of two proteins in HEK 293 cells is shown for red and green fluorescent proteins and also for Sox2 and Oct4

Independent inducible expression of two proteins in HEK 293 cells is shown for red and green fluorescent proteins (Panel A) and also for Sox2 and Oct4 (Panel B).

Lenti-X Packaging Single Shots (Envelope-Free) enable streamlined production of high-titer lentivirus pseudotyped with the envelope protein of choice by providing pre-aliquoted, lyophilized, single tubes of Xfect Transfection Reagent premixed with an optimized formulation of Lenti-X lentiviral packaging plasmids. The provided pEmpty Vector is used for cloning and expression of any envelope protein of choice (not supplied). Virus production simply involves reconstituting the lyophilized packaging mix with a lentiviral transfer vector of choice together with a plasmid encoding for the desired protein envelope in sterile water and adding it to 293T cells, e.g., Lenti-X 293T Cells (Cat. # 632180), in a 10-cm dish.

Cat. # 631294 consists of 16 tubes of Lenti-X Packaging Single Shots (Envelope-Free; Cat. # 631297), the pEmpty Vector (Cat. # 631298) for cloning and expression of the desired envelope protein, and the Lenti-X Reporter Vector Set (Cat. # 632677), which includes the self-inactivating transfer vectors encoding ZsGreen1 and firefly luciferase, respectively, under the CMV promoter, to be used as reporters for viral transduction.

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Workflow for pseudovirus production using Lenti-X Packaging Single Shots (Envelope-Free)

Workflow for pseudovirus production using Lenti-X Packaging Single Shots (Envelope-Free).

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631294: Lenti-X Packaging Single Shots (Envelope-Free)

Lenti-X SARS-CoV-2 Packaging Single Shots (B.1.351 Spike, Full Length) enable streamlined production of high-titer lentivirus pseudotyped with the spike protein from SARS-CoV-2 (B.1.351 variant) by providing pre-aliquoted, lyophilized, single tubes of Xfect Transfection Reagent premixed with an optimized formulation of Lenti-X lentiviral packaging plasmids. Pseudovirus production simply involves reconstituting a tube of lyophilized packaging mix with a lentiviral transfer vector of choice in sterile water and adding the mixture to 293T cells (e.g., Lenti-X 293T Cells, Cat. # 632180) in a 10-cm dish.

Each kit includes 12 tubes of Lenti-X SARS-CoV-2 Packaging Mix (B.1.351 Spike, Full Length; Cat. # 631290), 6 tubes of Lenti-X SARS-CoV-2 Packaging Mix (No-Envelope Control; Cat. # 632676) for production of pseudoviral particles lacking an envelope protein, and the Lenti-X SARS-CoV-2 Vector Set (Cat. # 632677), which includes two self-inactivating lentiviral transfer vectors encoding ZsGreen1 and firefly luciferase, respectively, under the CMV promoter, to be used as reporters for viral transduction.

For your convenience, the packaging mixes included in the kit are also sold separately.

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631292: Lenti-X SARS-CoV-2 Packaging Single Shots (B.1.351 Spike, Full Length)

Lenti-X SARS-CoV-2 Packaging Single Shots (B.1.351 Spike, Truncated) enable streamlined production of high-titer lentivirus pseudotyped with the spike protein from SARS-CoV-2 (B.1.351 variant) by providing pre-aliquoted, lyophilized, single tubes of Xfect Transfection Reagent premixed with an optimized formulation of Lenti-X lentiviral packaging plasmids. Pseudovirus production simply involves reconstituting a tube of lyophilized packaging mix with a lentiviral transfer vector of choice in sterile water and adding the mixture to 293T cells (e.g., Lenti-X 293T Cells, Cat. # 632180) in a 10-cm dish.

Each kit includes 12 tubes of Lenti-X SARS-CoV-2 Packaging Mix (B.1.351 Spike, Truncated; Cat. # 631291), 6 tubes of Lenti-X SARS-CoV-2 Packaging Mix (No-Envelope Control; Cat. # 632676) for production of pseudoviral particles lacking an envelope protein, and the Lenti-X SARS-CoV-2 Vector Set (Cat. # 632677), which includes two self-inactivating lentiviral transfer vectors encoding ZsGreen1 and firefly luciferase, respectively, under the CMV promoter, to be used as reporters for viral transduction.

For your convenience, the packaging mixes included in the kit are also sold separately.

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631293: Lenti-X SARS-CoV-2 Packaging Single Shots (B.1.351 Spike, Truncated)

Lenti-X SARS-CoV-2 Packaging Single Shots (Universal) allow streamlined production of high-titer lentivirus pseudotyped with your SARS-CoV-2 spike protein of interest by providing pre-aliquoted, lyophilized, single tubes of Xfect Transfection Reagent premixed with an optimized formulation of Lenti-X lentiviral packaging plasmids. Pseudovirus production simply involved diluting a SARS-CoV-2 spike variant expression vector and your transfer vector of choice in water, adding to a tube of single shots, and applying the mixture to 293T cells (e.g., Lenti-X 293T Cells, Cat. # 632180) in a 10-cm dish.

Each kit includes 16 tubes of Lenti-X Packaging Single Shots (Envelope-Free; Cat. # 631297), the pEmpty Vector (Cat. # 631298) for cloning and expression of the desired SARS-CoV-2 spike protein, the Lenti-X SARS-CoV-2 Spike Vector Set (Cat. # 631296), which includes three plasmids encoding for truncated SARS-CoV-2 spike variants D614G, B.1.1.7, and B.1.351 and the Lenti-X Reporter Vector Set (Cat. # 632677). Cat. # 632677 includes two self-inactivating lentiviral transfer vectors encoding ZsGreen1 and firefly luciferase, respectively, under the CMV promoter, to be used as reporters for viral transduction.

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Workflow for pseudovirus production using Lenti-X SARS-CoV-2 Packaging Single Shots (Universal)

Workflow for pseudovirus production using Lenti-X SARS-CoV-2 Packaging Single Shots (Universal).

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Transduction of an ACE2-positive cell line using pseudovirus produced with Lenti-X SARS-CoV-2 Packaging Single Shots (Universal)

Transduction of an ACE2-positive cell line with SARS-CoV-2 pseudovirus. Lenti-X Packaging Single Shots (Universal) was used to produce truncated SARS-CoV-2 spike B.1.1.7 pseudovirus encoding the fluorescent protein ZsGreen (Panel A) or SARS-CoV-2 spike B.1.1.7 pseudovirus encoding firefly luciferase (Panel B). Panel A. Concentrated supernatant (20X; 30 or 10 µl) was used to transduce a HEK293T cell line stably expressing the human ACE2 receptor in the presence of 6 µg/ml polybrene in 48-well plates. The transduction efficiencies for each sample were measured by flow cytometry 6 days post-transduction. The percentage of ZsGreen-positive (ZsGreen+) cells and mean fluorescence intensity (MFI) are shown. Panel B. Concentrated supernatant (20X; 30, 20, or 10 µl) was used to transduce a HEK293T cell line stably expressing the human ACE2 receptor in the presence of 6 µg/ml polybrene in 48-well plates. HEK293T cells lacking the ACE2 transgene were transduced to determine background luminescence levels (NC; negative control). Luminescence values for each sample were measured 6 days post-transduction.

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Lenti-X SARS-CoV-2 Packaging Single Shots (Universal) uses Takara Bio’s Lenti-X 4th-generation packaging system

Lenti-X Packaging Single Shots (Envelope-Free) uses Takara Bio’s Lenti-X 4th-generation packaging system. Panel A. Plasmids expressing all viral components except the SARS-CoV-2 spike protein that are contained in lyophilized Single Shots formulation. High expression of essential viral components are driven by the Tet-Off and Tat transactivators. In addition, the gag and pol genes are separated and the pol gene is fused to vpr to ensure transport of the reverse transcriptase/integrase protein into the recombinant viral particle. Panel B. The remaining viral component, the desired SARS-CoV-2 spike variant mutant is cloned into the pEmpty Vector and used in the single shots reaction. Expression of the spike variant mutant is driven by a CMV promoter.

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631295: Lenti-X SARS-CoV-2 Packaging Single Shots (Universal)

Highly concentrated and purified lentivirus for delivery of the Tet-On 3G transactivator to target cells.  Any gene of interest under the control of a TRE3G promoter can be tightly controlled by the presence and absence of Dox in the culture medium. Tet-On 3G is a modified, improved Tet-On Advanced transcriptional activator protein (transactivator) which has been evolved to display far higher sensitivity to the inducer molecule dox. Tet-On 3G generates very high maximal expression and responds to lower Dox concentrations than its predecessors.

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0055VCT: rLV.EF1.Tet3G-9

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Expression plasmid for inducible expression

Required Products

Cat. #	Product	Size	Price	License	Quantity	Details
631101	Tet System Approved FBS, US-Sourced	500 mL	USD $902.00
Fetal Bovine Serum (FBS) that has been functionally tested for optimal use with all of our Tet Systems. This FBS does not contain trace levels of tetracycline (or derivatives) which have been observed to interfere with proper regulation of TRE-controlled gene expression. Use of this serum ensures the maximal range of induction possible with the Tet Systems. This FBS has been processed in the United States. Documents Components Image Data Back Fetal bovine serum (FBS) approved for use with tetracycline-inducible expression systems Fetal bovine serum (FBS) approved for use with tetracycline inducible expression systems. Only Takara Bio USA functionally tests FBS to be sure that no contaminating tetracycline derivatives interfere with induced expression levels. Back Our fetal bovine serum is functionally tested to guarantee consistent results with tetracycline-inducible expression systems Fetal bovine serum from Clontech is functionally tested to guarantee consistent results with tetracycline-inducible expression systems. Serum from other vendors that have not been functionally tested may contain tetracyclines that affect the maximum expression in Tet-Off systems, or generate background in Tet-On systems. Data here shows induction of luciferase expression from our CHO-AA8-Luc Tet-Off Control Cell Line using different sources of FBS. Back 631101: Tet System Approved FBS, US-Sourced