The overall goal of this method is to enable depletion rescue experiments to be performed under conditions that preserve cellular integrity and protein homeostasis. For functional analyses of biological processes at the single cell level. This method can help answer key questions regarding protein functions and structure requirements in cell dynamics such as mitosis and cell differentiation.
The main advantage of this technique is to deplete a protein of interest and reintroduce at the same time variant of this protein at neo physiological level in a measurity of a cell population. Begin by coating glass bottom dishes with fribronectin. To do this, dilute a one milligram per milliliter firbronectin stock one to 100 with sterile PBS.
Add one milliliter to each 35 milliliter dish to cover the whole surface. Incubate for one hour at 37 degrees celsius and 5%carbon dioxide. During the incubation, pre-warm alpha MEM supplemented with 10%FBS and an aliquot of 0.05%trypsin EDTA to 37 degrees celsius.
After 45 minutes of incubation, start preparing the cell solution. Aspirate the medium from a 10 centimeter plate of HeLa RFP H2B cells. And gently rinse twice with 1.5 milliliters of 0.05%trypsin EDTA.
Leave 0.5 milliliters of trypsin EDTA on the plate after the last aspiration. And incubate the cells for two to three minutes at 37 degrees celsius. And 5%carbon dioxide.
Gently tap the plate. And observe the cell layer under the microscope to verify that all the cells have been detached. Then add 10 milliliters of medium and pipette gently several times to separate the cells and transfer the cell suspension in a 15 milliliter tube.
Count a 10 microliter sample of the cell suspension using a hemocytometer. Next, aspirate the fibronectin solution from the glass bottom dishes. Do not allow the fibronectin to dry before cell plating.
Prepare a cell suspension solution to plate 1.5 times ten to the fifth cells per 35 millimeter dish in two milliliters of medium. Plate one 35 millimeter plate additional to the number of conditions in order to count the cell number prior to virus transduction. Incubate for 30 to 45 minutes.
After the incubation time has elapsed, check the cells under the microscope to ensure that the cells are well separated. As seen here. Return the plates to the incubator and grow the cells until the next day.
On the day after plating, ensure that the cells have reached the density of at least 50%A cell density lower than 50%results in decreased virus transduction efficiency. Increased variation of protein expression per cell, and increased cell toxicity. Then harvest the cells from the additional plated dish as before.
And count the number of cells. Calculate the volume of viruses required for efficient transduction of the cells. Label a 1.5 milliliter microcentrifuge tube for each condition.
And add 400 microliters of warm alpha MEM minus. After slowly thawing aliquots of the viruses on ice, add the required volume of each virus to the tubes and mix gently by pipetting. Next, after aspirating the medium from the cells, gently add the virus mix drop wise.
Incubate the cells at 37 degrees celsius with 5%carbon dioxide. Every 15 minutes for a period of one hour, agitate the plates carefully under the sterile hood to cover the cells with virus. Using a small amount of medium facilitates the contact of the virus with the cells.
After the incubation, gently pipette 1.6 milliliters of alpha MEM minus to each plate to obtain a total volume of two milliliters. Then start cell synchronization by adding two millimolar thymidine before incubating the cells for a further two hours. Meanwhile, prepare the siRNA transfection mix by adding in a microtube 139 microliters of sterile water, 50 microliters of one molar calcium chloride, and 11 microliters of 20 micromolar siRNA.
If no siRNA is necessary, replace the amount of siRNA by sterile water to perform an empty transfection. Add HBSS2X solution drop wise slowly on to the calcium chloride siRNA mixture. Gently mix two times by air injection using a 200 microliter pipette.
As smaller precipitates result in better transfection efficiency. Then incubate the mix for 30 minutes at room temperature. After the incubation, slowly add 200 microliters drop wise to each plate and agitate to mix.
Incubate the plates at 37 degrees celsius, 5%carbon dioxide for 16 hours. The next day, remove the dishes from the incubator and transport them to a tissue culture hood. After rinsing the cells twice with two milliliters of warm hipis, add two milliliters of alpha MEM minus before returning to the incubator.
Seven hours later, add two millimolar thymidine to the plates before returning them to the incubator. Work with no more than four cell plates at a time as temperature variations effect the length of the cell cycle. The following day, 48 hours after siRNA transfection and virus infection, rinse the cells twice with two milliliters of warm PBS then add two milliliters of alpha MEM without phenol red for live cell imaging.
Incubate for seven hours. Perform short term live cell imaging experiments on mitotic cells with an inverted microscope equipped with a humidified 5%carbon dioxide thermo regulated chamber. Prior to acquisition, verify that the chamber has reached the appropriate temperature of 37 degrees celsius.
Place the cultured dishes in the microscope chamber one hour before acquisition to enable proper equilibrium of the medium and avoid focus drifting due to temperature changes. Examine the mitotic status of the cells. At this point.
10 to 15%of the cells should be in the early stages of mitosis. During the equilibration time, set up the acquisition parameters as determined in prior experiments. It is important to achieve appropriate resolution without photo bleaching.
As this causes cell damage and perturbs mitotic progression. Choose several fields containing cells that are at mitotic entry per condition to obtain a significant number of cells to analyze without exceeding the acquisition interval. Once all fields have been chosen, reset the focus and start the acquisition as fast as possible.
Using a spinning disc can focus system. A typical set up would include four different conditions. Seven fields per plate.
And two color channel with a 1.5 to two minute interval over 75 minute period. Determine well defined criteria to analyze mitotic pheno types in cells which will depend on the fluorescent markers being used. In contrast to transfection of BAG3 GFP, plasmic DNA, as seen on the left, adenofection with recompetent adenoviruses shown here on the right enables a more homogeneous and efficient expression of BAG3 GFP.
In a majority of the cell population at low multiplicity of infection. This representative western blot shows that adenofection with BAG3 specific siRNAs and recompotent adenovirus BAG3 GFP allows efficient depletion of endogenous BAG3 and reintroduction of the BAG3 GFP protein at near endogenous levels. As shown here, adenofection can preserve protein homeostasis since there is no detectable increase in the levels of the stress induced heat shock proteins in HeLa cells that have been adenofected.
In contrast to HeLa cells treated with the proteotoxic stress inducer, MG132. Here we see that the progression of cells through mitosis is not significantly perturbed by adenofection as shown by these con focal time lapse images from HeLa cells adenofected with control siRNA and BacMam GFP alpha Tubulin. And BacMam FRP Actin.
The graph indicates the proportion of cells with abnormal mitosis. Cells adenofected with BAG3 specific siRNA alone exhibited an approximate two fold increase in the level of mitotic defects. As depicted by the red bar.
Which was restored to near the control cell level by reintroduction of BAG3 GFP shown by the black bar. After watching this video, you should have a good understanding on how to perform depletion rescue experiments using adenofection. This method provides a fast and simple system to create hypomorphic knock downs for structure function analysis in multiple cellular backgrounds.
Following this method, key structural requirements on the protein of interest for a given function can be identified. Other approaches of genome editing can then be used to deepen the mechanics involved.
