Lentiviral particles for fluorescent labeling of specific subcellular structures

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Map schematics of expression cassettes contained in particles for labeling subcellular compartments (e.g. actin, tubulin, mitochondria, etc.) with mCherry red fluorescent protein

Map schematics of expression cassettes contained in particles for labeling subcellular compartments (e.g. actin, tubulin, mitochondria, etc.) with mCherry red fluorescent protein.

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Fluorescent images of mammalian cells transduced with either AcGFP1-Mem (left) or mCherry-Mem (right) virus showing clear labeling of the plasma membrane

Fluorescent images of mammalian cells transduced with either AcGFP1-Mem (left) or mCherry-Mem (right) virus showing clear labeling of the plasma membrane. (Cat. # 0019VCT and 0026VCT, respectively.) The blue channel in the right image is DAPI staining of the nucleus.

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0026VCT: rLV.EF1.mCherry-Mem-9

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Fluorescent labelling of endoplasmic reticulum (ER)

Fluorescent labelling of endoplasmic reticulum (ER). Cells transduced with this premade lentivirus (Cat. No. 0025VCT) will express a fusion protein consisting of mCherry fluorescent protein; the ER targeting sequence of calreticulin, which is fused at the 5' end; and the sequence encoding the ER retrieval sequence, KDEL, which is fused at the 3' end.

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Map schematics of expression cassettes contained in particles for labeling subcellular compartments (e.g. actin, tubulin, mitochondria, etc.) with mCherry red fluorescent protein

Map schematics of expression cassettes contained in particles for labeling subcellular compartments (e.g. actin, tubulin, mitochondria, etc.) with mCherry red fluorescent protein.

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0025VCT: rLV.EF1.mCherry-ER-9

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Visualizing mitochondria in mammalian cells using mCherry or AcGFP1 fluorescent proteins

Visualizing mitochondria in mammalian cells using mCherry or AcGFP1 fluorescent proteins. HCT116 cells and Hela cells were transduced using Cat. Nos. 0024VCT and 0017VCT respectively. The lentivirus vectors express a fusion protein consisting of a fluorescent protein tagged with the targeting sequence from subunit VIII of cytochrome c oxidase that targets mitochondria.

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Map schematics of expression cassettes contained in particles for labeling subcellular compartments (e.g. actin, tubulin, mitochondria, etc.) with mCherry red fluorescent protein

Map schematics of expression cassettes contained in particles for labeling subcellular compartments (e.g. actin, tubulin, mitochondria, etc.) with mCherry red fluorescent protein.

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0024VCT: rLV.EF1.mCherry-Mito-9

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The nuclei of HCT116 cells visualized using recombinant lentivirus expressing AcGFP1 or mCherry fluorescent proteins fused to a nuclear localization signal (Cat. Nos. 0016VCT or 0023VCT, respectively)

The nuclei of HCT116 cells visualized using recombinant lentivirus expressing AcGFP1 or mCherry fluorescent proteins fused to a nuclear localization signal (Cat. Nos. 0016VCT or 0023VCT, respectively).

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Map schematics of expression cassettes contained in particles for labeling subcellular compartments (e.g. actin, tubulin, mitochondria, etc.) with mCherry red fluorescent protein

Map schematics of expression cassettes contained in particles for labeling subcellular compartments (e.g. actin, tubulin, mitochondria, etc.) with mCherry red fluorescent protein.

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0023VCT: rLV.EF1.mCherry-Nuc-9

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Map schematics of expression cassettes contained in particles for labeling subcellular compartments (e.g. actin, tubulin, mitochondria, etc.) with mCherry red fluorescent protein

Map schematics of expression cassettes contained in particles for labeling subcellular compartments (e.g. actin, tubulin, mitochondria, etc.) with mCherry red fluorescent protein.

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0022VCT: rLV.EF1.mCherry-Golgi-9

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Fluorescent labelling of tubulin in mammalian cells

Fluorescent labelling of tubulin in mammalian cells. HeLa cells were infected at 80 M.O.I. with ready-made lentivirus engineered to expressed AcGFP1-Tubulin (Right image; Cat. No. 0014VCT) or mCherry Tubulin (Left image; Cat. No. 0021VCT).

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Map schematics of expression cassettes contained in particles for labeling subcellular compartments (e.g. actin, tubulin, mitochondria, etc.) with mCherry red fluorescent protein

Map schematics of expression cassettes contained in particles for labeling subcellular compartments (e.g. actin, tubulin, mitochondria, etc.) with mCherry red fluorescent protein.

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0021VCT: rLV.EF1.mCherry-Tubulin-9

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Subcellular staining of actin using recombinant lentivirus to deliver and express fusions of actin to mCherry (Cat. # 0020VCT rLV.EF1.mCherry-Actin-9) and AcGFP1 (Cat. # 0013VCT rLV.EF1.AcGFP1-Actin-9), respectively

Subcellular staining of actin using recombinant lentivirus to deliver and express fusions of actin to mCherry (Cat. # 0020VCT rLV.EF1.mCherry-Actin-9) and AcGFP1 (Cat. # 0013VCT rLV.EF1.AcGFP1-Actin-9), respectively.

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Not all titers are the same! Comparison of cells transduced with 100 M

Not all titers are the same! Comparison of cells transduced with 100 M.O.I. of lentivirus provided by Clontech/Vectalys versus 100 M.O.I. of lentivirus provided by Vendor S. Each premade virus preparation expresses a green fluorescent protein when transduced. Clontech/Vectalys provides a functional titer, so every cell treated with our virus preparation was transduced with multiple copies of virus, leading to strong expression of the fluorescent protein. By contrast, the titer provided by Vendor S includes nonfunctional lentivirus particles as a large percentage of the titer so 100 M.O.I of their virus preparation is not sufficient to effectively transduce the cells.

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Map schematics of expression cassettes contained in particles for labeling subcellular compartments (e.g. actin, tubulin, mitochondria, etc.) with mCherry red fluorescent protein

Map schematics of expression cassettes contained in particles for labeling subcellular compartments (e.g. actin, tubulin, mitochondria, etc.) with mCherry red fluorescent protein.

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0020VCT: rLV.EF1.mCherry-Actin-9

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Map schematics of expression cassettes contained in particles for labeling subcellular compartments (e.g. actin, tubulin, mitochondria, etc.) with AcGFP1 fluorescent protein

Map schematics of expression cassettes contained in particles for labeling subcellular compartments (e.g. actin, tubulin, mitochondria, etc.) with AcGFP1 fluorescent protein.

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Fluorescent images of mammalian cells transduced with either AcGFP1-Mem (left) or mCherry-Mem (right) virus showing clear labeling of the plasma membrane

Fluorescent images of mammalian cells transduced with either AcGFP1-Mem (left) or mCherry-Mem (right) virus showing clear labeling of the plasma membrane. (Cat. # 0019VCT and 0026VCT, respectively.) The blue channel in the right image is DAPI staining of the nucleus.

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0019VCT: rLV.EF1.AcGFP1-Mem-9

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Endosome labeling using concentrated and purified lentivirus

Endosome labeling using concentrated and purified lentivirus. HeLa cells were transduced using rLV.EF1.AcGFP1-Endo-9 (Cat. No 0018VCT) at an M.O.I of 80. The ready-made lentivirus encodes a fusion of RhoB GTPase and AcGFP1 which, when expressed, localizes to the surface of vesicles of the endocytic pathway.

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Map schematics of expression cassettes contained in particles for labeling subcellular compartments (e.g. actin, tubulin, mitochondria, etc.) with AcGFP1 fluorescent protein

Map schematics of expression cassettes contained in particles for labeling subcellular compartments (e.g. actin, tubulin, mitochondria, etc.) with AcGFP1 fluorescent protein.

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0018VCT: rLV.EF1.AcGFP1-Endo-9

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Visualizing mitochondria in mammalian cells using mCherry or AcGFP1 fluorescent proteins

Visualizing mitochondria in mammalian cells using mCherry or AcGFP1 fluorescent proteins. HCT116 cells and Hela cells were transduced using Cat. Nos. 0024VCT and 0017VCT respectively. The lentivirus vectors express a fusion protein consisting of a fluorescent protein tagged with the targeting sequence from subunit VIII of cytochrome c oxidase that targets mitochondria.

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Map schematics of expression cassettes contained in particles for labeling subcellular compartments (e.g. actin, tubulin, mitochondria, etc.) with AcGFP1 fluorescent protein

Map schematics of expression cassettes contained in particles for labeling subcellular compartments (e.g. actin, tubulin, mitochondria, etc.) with AcGFP1 fluorescent protein.

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0017VCT: rLV.EF1.AcGFP1-Mito-9

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The nuclei of HCT116 cells visualized using recombinant lentivirus expressing AcGFP1 or mCherry fluorescent proteins fused to a nuclear localization signal (Cat. Nos. 0016VCT or 0023VCT, respectively)

The nuclei of HCT116 cells visualized using recombinant lentivirus expressing AcGFP1 or mCherry fluorescent proteins fused to a nuclear localization signal (Cat. Nos. 0016VCT or 0023VCT, respectively).

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Map schematics of expression cassettes contained in particles for labeling subcellular compartments (e.g. actin, tubulin, mitochondria, etc.) with AcGFP1 fluorescent protein

Map schematics of expression cassettes contained in particles for labeling subcellular compartments (e.g. actin, tubulin, mitochondria, etc.) with AcGFP1 fluorescent protein.

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0016VCT: rLV.EF1.AcGFP1-Nuc-9

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The golgi apparatus of HCT116 cells visualized using recombinant lentivirus (Cat. No. 0015 VCT rLV.EF1.AcGFP1-Golgi-9) expressing AcGFP1 fluorescent protein fused to the N-terminal 81 amino acids of human β 1,4-galactosyltransferase, which contains a membrane-anchoring signal peptide

The golgi apparatus of HCT116 cells visualized using recombinant lentivirus (Cat. No. 0015 VCT rLV.EF1.AcGFP1-Golgi-9) expressing AcGFP1 fluorescent protein fused to the N-terminal 81 amino acids of human β 1,4-galactosyltransferase, which contains a membrane-anchoring signal peptide.

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Map schematics of expression cassettes contained in particles for labeling subcellular compartments (e.g. actin, tubulin, mitochondria, etc.) with AcGFP1 fluorescent protein

Map schematics of expression cassettes contained in particles for labeling subcellular compartments (e.g. actin, tubulin, mitochondria, etc.) with AcGFP1 fluorescent protein.

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0015VCT: rLV.EF1.AcGFP1-Golgi-9

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Fluorescent labelling of tubulin in mammalian cells

Fluorescent labelling of tubulin in mammalian cells. HeLa cells were infected at 80 M.O.I. with ready-made lentivirus engineered to expressed AcGFP1-Tubulin (Right image; Cat. No. 0014VCT) or mCherry Tubulin (Left image; Cat. No. 0021VCT).

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Map schematics of expression cassettes contained in particles for labeling subcellular compartments (e.g. actin, tubulin, mitochondria, etc.) with AcGFP1 fluorescent protein

Map schematics of expression cassettes contained in particles for labeling subcellular compartments (e.g. actin, tubulin, mitochondria, etc.) with AcGFP1 fluorescent protein.

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0014VCT: rLV.EF1.AcGFP1-Tubulin-9

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Various cell types can be transduced with Vectalys lentiviral particles, including primary and stem cell lines

Various cell types can be transduced with Vectalys lentiviral particles, including primary and stem cell lines. The suggested seeding concentration and MOI vary depending on the specific cell type to be transduced. When the recommendations in this table are followed, transduction efficiency is high for immortalized, primary, and stem cells, regardless of whether they are adherent or suspension cells.

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Overview of the process for generating high-titer lentiviral particles

Overview of the process for generating high-titer lentiviral particles. Through this proprietary, large-scale process Vectalys is able to produce very high-titer, high-purity lentiviruses ready for transduction of many different cell types. The high titer allows for manipulation of even hard-to-transduce cells, like primary cells or stem cells, and the exceptional purity prevents cytotoxicity.

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Comparison of the purity of lentiviruses generated with Vectalys’ method or a classic purification method

Comparison of the purity of lentiviruses generated with Vectalys’ method or a classic purification method. Lentiviral proteins were separated by SDS-PAGE and stained with Coomassie Blue. The high purity of Vectalys lentiviral particles is clear when comparing the two methods; even when Vectalys particles are loaded at a 10-fold higher amount, very little contaminating protein is seen. The ratio of total particles (physical particles) to functional particles (transducing units) is lower for Vectalys purified lentivirus compared to classic methods of purification, indicating higher purity. Data provided by Vectalys.

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Subcellular staining of actin using recombinant lentivirus to deliver and express fusions of actin to mCherry (Cat. # 0020VCT rLV.EF1.mCherry-Actin-9) and AcGFP1 (Cat. # 0013VCT rLV.EF1.AcGFP1-Actin-9), respectively

Subcellular staining of actin using recombinant lentivirus to deliver and express fusions of actin to mCherry (Cat. # 0020VCT rLV.EF1.mCherry-Actin-9) and AcGFP1 (Cat. # 0013VCT rLV.EF1.AcGFP1-Actin-9), respectively.

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Not all titers are the same! Comparison of cells transduced with 100 M

Not all titers are the same! Comparison of cells transduced with 100 M.O.I. of lentivirus provided by Clontech/Vectalys versus 100 M.O.I. of lentivirus provided by Vendor S. Each premade virus preparation expresses a green fluorescent protein when transduced. Clontech/Vectalys provides a functional titer, so every cell treated with our virus preparation was transduced with multiple copies of virus, leading to strong expression of the fluorescent protein. By contrast, the titer provided by Vendor S includes nonfunctional lentivirus particles as a large percentage of the titer so 100 M.O.I of their virus preparation is not sufficient to effectively transduce the cells.

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Map schematics of expression cassettes contained in particles for labeling subcellular compartments (e.g. actin, tubulin, mitochondria, etc.) with AcGFP1 fluorescent protein

Map schematics of expression cassettes contained in particles for labeling subcellular compartments (e.g. actin, tubulin, mitochondria, etc.) with AcGFP1 fluorescent protein.

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What is a functional titer? Most vendors of ready-made viral particles determine their titers using a simple particle count

What is a functional titer? Most vendors of ready-made viral particles determine their titers using a simple particle count. This count will include free capsid protein, nonfunctional and immature virions, as well as functional virus.The functional titer provided by Clontech and Vectalys is not just the total particle number, it is a measurement of the total number of virions capable of integrating into cells, measured by transducing HCT116 cells. In real terms, this means our viral preps contain up to 100-fold more infectious particles than preps from other vendors.

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Achieve 100% transduction efficiency even in cell types that are typically hard to infect

Achieve 100% transduction efficiency even in cell types that are typically hard to infect. Super-concentrated highly purified lentivirus prepared by Vectalys permits transduction at high M.O.I.

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0013VCT: rLV.EF1.AcGFP1-Actin-9