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Investigación en Ciencias de la Vida
 

Lentiviral Biosensors

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Lentiviral Biosensors for GFP- & RFP-LC3 and p62

The GFP-LC3 displays a diffuse nuclear and cytosolic distribution in fed cells, and a punctate distribution in starved autophagic cells.
HeLa cells were transduced with LentiBrite™ GFP-LC3 lentiviral particles and were either (A) left in complete media or (B) incubated in EBSS containing a lysosome inhibitor under starvation conditions. The GFP-LC3 displays a diffuse nuclear and cytosolic distribution in fed cells, and a punctate distribution in starved autophagic cells. (Click to enlarge)
Biosensors can be used to detect a particular protein as well as the subcellular location of that protein within live cells. Fluorescent tags are an effective way to visualize the protein of interest within a cell by either fluorescent microscopy or time-lapse video capture. Visualizing live cells without disruption can reveal changing cellular conditions in real time.

Lentiviral vector systems are a popular tool for introducing genes and gene products into cells. Advantages over non-viral methods (such as chemical-based transfection) include higher-efficiency transfection of dividing and non-dividing cells, stable expression of the transgene, and low immunogenicity.

Merck's new LentiBrite™Lentiviral Biosensors, a new suite of pre-packaged lentiviral particles encoding foundational proteins of autophagy detection - LC3 & p62, enabling precise visualization of autophagosome formation under different cell/disease states in live cell and in vitro analysis. Visualize autophagy in real time, even in difficult-to-transfect cell types, using LentiBrite GFP- & RFP-tagged LC3 & p62 wild-types and LC3-G120A mutant control lentiviral biosensors.

LentiBrite Biosensor Advantages:

  • Pre-packaged, ready-to-use, fluorescently-tagged LC3 & p62 with monomeric GFP & RFP
  • Minimum titer (≥3 x 10^8 IFU/mL) per vial
  • Long-term, stable fluorescent expression that is non-disruptive towards cellular function
  • Higher efficiency transfection as compared to traditional chemical-based and other non-viral-based transfection methods
  • Ability to transfect dividing, non-dividing, and difficult-to-transfect cell types, such as primary cells or stem cells
  • Validated for fluorescent microscopy and live cell analysis
  • LC3 Control Mutant lentiviral particle contains the translocation-defective protein LC3-G120A for comparison studies.

DescriptionCatalogue No.
LentiBrite GFP-LC3 Lentiviral Biosensor 17-10193
LentiBrite RFP-LC3 Lentiviral Biosensor 17-10143
LentiBrite GFP-LC3 Control Mutant Lentiviral Biosensor 17-10189
LentiBrite RFP-LC3 Control Mutant Lentiviral Biosensor 17-10188
LentiBrite GFP-p62 Lentiviral Biosensor 17-10224
LentiBrite RFP-p62 Lentiviral Biosensor 17-10404


Visualization of Autophagy

Visualizing LC3 & p62 localization from a diffuse distribution in non-autophagic conditions.
Timelapse Fluorescence Imaging:
U2OS cells were plated in coverglass chamber slides and transduced with GFP-p62 lentiviral particles. Images were collected every 20 seconds for a total of 32 min. Shown here are 3 sequential frames. Cells were left in complete media or incubated in EBSS containing a lysosome inhibitor to induce autophagosome formation and inhibit lysosomal degradation. The GFP-p62 displays a diffuse cytosolic distribution in fed cells (A), and a punctate distribution in starved autophagic cells (B,C). (Click image to enlarge.)
Visualizing LC3 & p62 localization from a diffuse distribution in non-autophagic conditions to a punctate distribution in autophagic cells is the primary tool used by researchers to study the formation of the autophagasome and thus, macroautophagy. Transduction of a fluorescently-tagged LC3 or p62 is not only the best way to detect LC3 & p62 integration into the autophagosome using fluorescent microscopy, but also provides the researcher the ability to monitor this event using live cell or time-lapse visual analysis.

Of the many ways in which GFP- or RFP-LC3 and p62 can be transduced into a cell, lentiviral delivery is the most efficient and effective. Lentiviral transfection is capable of introducing the GFP- or RFP-LC3 and p62 constructs into more a larger number of cell types as compared to baculovirus or chemical transfection, is capable of transfecting primary cells, and shows comparable or superior expression to antibody staining.

Merck’s LentiBrite GFP- & RFP-LC3 and p62 lentiviral particles allow users to easily and confidently transfect their cells of choice to visualize LC3 or p62 localization within the autophagosome.

LentiBrite vs. Antibody Comparison and Inhibitor Analysis:

LentiBrite vs. antibody comparison and inhibitor analysis.
Fluorescence Microscopy
HeLa cells were plated in a chamber slide and transduced with LentiBrite RFP-LC3 lentiviral particles at an MOI of 40 for 24 hours. After media replacement and 48 hours further incubation, cells were either (A) left in complete media, (B) incubated for 4 hours in EBSS containing a lysosome inhibitor to induce autophagy and inhibit lysosomal degradation, or (C) incubated as in (B), with the addition of 5 mM 3-methyladenine (3-MA) as an inhibitor of autophagy. 3-MA completely blocks formation of RFP-LC3-positive autophagic punctae. Immunocytochemical staining (green) of the same fields of view with a monoclonal antibody against LC3A reveals similar expression patterns (D, E, F) as compared to the virally-transduced RFP-protein (red). (Click image to enlarge.)


Higher Efficiency Transfection Compared to Baculovirus

MOI = 20
LentiBrite
Merck:/Freestyle/BI-Bioscience/Antibodies-Assays/cancer-images/transfection-lentibrite.jpg		 Baculovirus Competitor Product
Primary cell type HUVEC were plated in chamber slides and transduced with lentiviral particles at an MOI of 40 for 24 hours.
LentiBrite lentiviral particles encoding GFP-LC3 (Catalogue No. 17-10193) were adjusted to 1 mL, and equivalent volumes of lentivirus and baculovirus from a competitor product were added to HT1080 cells at an MOI of 20. Lentiviral transfection provides a stronger overall signal of GFP-LC3 compared to baculovirus transfection, when using equivalent amounts of each product. Click image to enlarge.


Ability to Transfect Dividing, Non-dividing, and Difficult-to-Transfect Cell Types

induceing autophagy and inhibiting lysosomal degradation of autophagosomes. Primary cell type HUVEC were plated in chamber slides and transduced with lentiviral particles at an MOI of 40 for 24 hours. Subsequent treatments for (A) cells left in complete media or (B) cells incubated in EBSS with lysosome inhibitor were performed.
Merck:/Freestyle/BI-Bioscience/Antibodies-Assays/cancer-images/rfp-lc3-lentiviral-biosensor.jpg Primary cell type, Human mesenchymal stem cells (HuMSC), were plated in a chamber slide and transduced with lentiviral particles at an MOI of 20 for 24 hours. After media replacement and 48 hours further incubation, cells were either (A) left in complete media or (B) incubated for 4 hours in EBSS containing a lysosome inhibitor, to induce autophagy and inhibit lysosomal degradation of autophagosomes.

Cells were fixed with formaldehyde and mounted. Images were obtained by oil immersion wide-field fluores-cence microscopy. The RFP-LC3 displays a diffuse cytosolic distribution in fed cells, and a punctate distribution in starved autophagic cells.

Live Cell Imaging of Autophagy

The introduction of genetically-encoded fluorescent protein fusions as a localization marker in living cells has revolutionized the field of cell biology. The ability to visualize and track LC3 localization to the autophagosome with spatial and temporal resolution is important to understanding the autophagic process.

Merck’s LentiBrite GFP- & RFP-LC3 lentiviral particles provide stable expression of the fluorescently-encoded transgene for reliable visualization of LC3 distribution over time, allowing for live cell imaging and analysis.

LentiBrite GFP-LC3 Lentiviral Biosensor (Catalogue No. 17-10193)

Timecourse imaging of HT-1080 cells after transduction with LentiBrite GFP-LC3 and the addition of a lysosomal inhibitor under starvation conditions. GFP-LC3 displays a diffuse nuclear and cytosolic distribution in fed cells, and a punctate distribution in starved, autophagic cells.