培養昆虫細胞における一過性発現および組換えタンパク質の細胞内局在

The overall goal of this procedure is to express fluorescently tagged proteins of interest within commercially available insect cells for the enhanced elucidation of protein function and or cellular trafficking of proteins. This method can help answer key cell biology and biochemistry based questions regarding protein functionality, protein interactions, and or subcellular protein localization. The main advantage of this technique is that constructs can be rapidly generated and expressed proteins functionally assessed in live cells without deleterious effects associated with baculovirus expression, thus improving throughput.

Though this method can provide insight in the study of insect proteins, it can also be applied to any system for which the study of protein function or cellular localization is desired. Demonstrating the procedure for insect cell culture and transfection will be Danni LeRoy, a technician from my laboratory. To begin this procedure, remove stock vials of frozen SF9 and Tni cells from the minus 80 degrees celsius freezer and allow them to thaw in a 37 degree celsius water bath.

After thawing, decontaminate the vials with 70%ethanol and place them on ice. All cell manipulations requiring the opening of vials and tissue culture flasks must be performed within a laminar flow hood to maintain sterile conditions. Add four milliliters of serum-free insect medium to a new T25 flask and four milliliters of TNM-FH medium to another T25 flask.

Transfer one milliliter of thawed Tni cells to the flask with serum-free insect medium and one milliliter of thawed SF9 cells to the flask with TNM-FH medium. Place the flasks in a 28 degree celsius non-humidified incubator and allow the cells to attach for 30 to 45 minutes. Replace the seeding medium with five milliliters of the appropriate medium.

And return the flasks to the 28 degree celsius non-humidified incubator. Monitor cell confluence daily. Passage the cells when they reach 90%confluency as shown by these representative images.

Tilt the flask containing the confluent cells so that the medium flows toward one corner away from the cell monolayer and use a sterile five milliliter serological pipette to carefully remove the medium without disturbing the cells. For Tni cells, use a new sterile five milliliter serological pipette to gently add four milliliters of serum-free insect medium onto the confluent monolayer. Move the pipette tip across the flask and slowly irrigate to remove cells loosely attached to the flask bottom.

To check for the adequate detachment of cells, remove all media, turn the flask over and observe that the bottom of the flask is clear. For SF9 cells, which adhere more tightly, add four milliliters of fresh TNM-FH medium and use a cell scraper to dislodge the attached cells. Use a five milliliter serological pipette to gently mix and reduce cell clumping.

After detaching the cells, transfer approximately 0.1 milliliters of each cell and medium mixture to a 1.5 milliliter micro-fuge tube. In a separate 0.5 milliliter micro-fuge tube add 10 microliters of each cell and medium mixture to 10 microliters of trypan blue. Remove the cell counter chamber slide from its packaging and add 10 microliters of the cell medium trypan blue mixture to each side of the counting slide.

Insert the slide into an automated cell counter and determine the cell density and viability. Transfer approximately one to 1.5 times 10 to the 6th cells to T25 flasks with fresh media. Label the flasks with the cell line, date, medium used, number of cells added, and passage number.

Place the flasks in a 28 degree celsius incubator for up to 72 hours. The procedure for insect cell transfection must also be performed within a laminar flow hood. Seed a T25 flask with up to one times 10 to the 6th Tni or SF9 cells in an appropriate insect cell medium.

Tni cells are used in this demonstration. Grow the cells to confluency for 72 hours at 18 degrees celsius. 72 hours later, remove and discard the old medium and dislodge the Tni cells with four milliliters of fresh serum-free insect medium as shown earlier.

The most difficult aspect of this procedure is obtaining the appropriate density of cells attached to the glass bottom dishes during transfection. No more than seven times 10 to the 5th cell should be added to each dish. After using an automated cell counter to estimate the cell density, thoroughly but gently mix the cell suspension by inverting the tube and add approximately seven times 10 to the 5th cells to individual 35 millimeter glass bottom dishes.

Allow the cells to attach for 20 to 25 minutes at 28 degrees celsius. For each transfection, add two micrograms of plasmid DNA to 0.1 milliliters of serum-free insect medium without FBS in a sterile 1.5 milliliter micro-fuge tube. In a separate tube, mix eight microliters of transfection reagent with 0.1 milliliters of serum-free insect medium.

Then transfer the solution to the tube containing the plasmid DNA of interest. Lightly vortex and incubate at room temperature for 20 to 30 minutes. Next, dilute the plasmid transfection mixture with 0.8 milliliters of serum-free insect medium, bringing the total volume up to one milliliter.

Carefully remove the media from the glass dish containing attached cells. Overlay the attached cells with the diluted plasmid transfection medium. Incubate the cells at 28 degrees celsius for five hours.

After five hours, remove and discard the transfection medium and gently wash the cells with one milliliter of serum-free insect medium, being careful not to dislodge the cells. Add two milliliters of fresh serum-free insect cell medium and incubate at 28 degrees celsius for 48 to 72 hours. 48 to 72 hours after transfection of the insect cells, wash the cells once with one milliliter of IPL-41 insect medium and then cover with two milliliters of IPL-41 for imaging.

Add four drops of Hoechst live cell staining reagent to the medium and incubate at 28 degrees celsius for 20 to 25 minutes. Place the 35 millimeter dish into the self enclosed laser scanning confocal microscope. Although many glass dishes are commercially available, it's important that they fit the microscope stage and it may be necessary to empirically determine which glassware is most compatible with cell attachment.

Adjust the microscope for Hoechst, EGFP and mCherry observation conditions. 359 nanometers and 461 nanometers for Hoechst excitation and emission. 489 nanometers and 510 nanometers for EGFP excitation and emission.

And 580 nanometers and 610 nanometers for mCherry excitation and emission. Using a 10X objective, perform an initial scan to confirm fluorescent expression. After that, switch to scanning mode using a 60X phase contrast water immersion objective.

Adjust the laser power, detector sensitivity, scanning speed, Z-axis depth, and digital zoom to optimize the image contrast and resolution. Image the cells at 1.5X digital zoom to give a total of 90X amplification. Save and export the raw data as TIFF image files for later analysis.

Tni cells were transfected with the indicated plasmids and the expression of recombinant proteins was visualized using confocal fluorescence microscopy. Successful transfection and expression of recombinant BtDrip1-EGFP is evident by the presence of green fluorescence on the cell's surface. Cells transfected with BtDrip2 version one EGFP show green fluorescence within indicating the intracellular expression of BtDrip2 version one.

Likewise, cells transfected with PIB DmSPR-mCherry or PIB PLA2G15-mCherry show red fluorescence indicating the expression of the respective chimeras. In the merged images, the orange or yellow areas indicate expression of both EGFP and mCherry which suggests that the proteins are co-localized within the same subcellular structures. An overlay of cells double transfected with PIB BtDrip1-EGFP and PIB DmSPR-mCherry suggests co-localization of BtDrip1-EGFP and DmSPR-mCherry on the cell's surface.

Cells double transfected with BtDrip2 version one EGFP and PIB DmSPR-mCherry show little co-localization of green and red fluorescence signals confirming the intracellular expression of BtDrip2 version one. In contrast the co-expression of PIB BtDrip2 version one EGFP and the PIB PLA2G15-mCherry lysosomal marker resulted in a significant overlap in the cytoplasmic green and red fluorescent signals. This strongly suggests that BtDrip2 is traffic to intercellular lysosomes.

After watching this video, you should have a good understanding of how to express fluorescent protein chimeras within cultured insect cells. This system offers rapid vector construction in protein synthesis, avoids challenges of virus based expression systems, and provides a robust means to observe cellular trafficking proteins.