The overall goal of this procedure is to measure the kinetics of mRNA nuclear export in mammalian cells. This is achieved by first micro injecting mRNA or DNA into the nuclei of mammalian tissue culture cells. Following microinjection, the samples are fixed after various incubation periods.
The mRNA is then stained by fluorescent in situ hybridization or fish. The final step of the procedure is to image the cells and quantify the amount of fluorescence in the cytoplasm and nucleus. Ultimately, the rate of mRNA export can be measured through a combination of microinjection and fluorescent microscopy.
Hi, I'm SRG AV from the laboratory of Alex Palazzo in the Department of Biochemistry at the University of Toronto. I'm Stefan Toski, also from the Palazzo Lab. I am Amy from the Palazzo lab and I'm Khi Maha David from the Palazzo lab.
Today we will show you a procedure for introducing mRNA into the nuclei of mammalian tissue culture cells by microinjection. We will also demonstrate how to stain the mRNA by fluorescent in C two hybridization. We use these procedures in our laboratory to study the kinetics of mRNA nuclear export in mammalian cells.
So let's get started. Prior to microinjection dute, previously prepared plasma DNA to between 50 and 200 micrograms per milliliter in injection buffer. Next at Oregon Green 4 88 or OG conjugated to 70 kilodalton dextran to the mixture to enable the identification of injected cells.
The OG conjugated dextran will help determine how much of the fluid was injected into the nucleus and how much leaked into the cytoplasm centrifuge. The samples at 13, 000 G and four degrees Celsius for at least 20 minutes to pellet any particulate matter that may potentially obstruct the needle tip. Choose the mammalian cell line to be micro injected based on the criteria described in the written protocol cost.
Seven cells are used for this demonstration seed. The cells on acid washed cover slips in 30 millimeter Petri dishes at least 24 hours prior to microinjection. Ideally, the cell monolayer should be roughly 70 to 90%confluent at the time of injection to allow for easy identification of the injected cells, the cell monolayer is wounded prior to injection by using a 200 microliter plastic pipette tip.
Etch a cross wound on the cover slip and leave the cells to recover in the tissue culture incubator for at least 15 minutes prior to injection. For microinjection, use an inverted microscope that is isolated on an air table to minimize vibrations that can be disrupted to the microinjection process. Using an einor gel loader tip, remove approximately one microliter from the top of the centrifuge sample to avoid disrupting the pellet.
Insert the tip of the pipette into the backend of a prefabricated microinjection needle and eject the liquid. The liquid will be drawn into the needle tip by capillary action. A meniscus near the tip should be visible within five to 30 seconds.
Place the fluid-filled needle into the wand and clamp it to the micro manipulator. At a 45 degree angle, visualize the needle using the 10 x objective and position it in the center of the viewing field near the focal plane. Using the coarse micro manipulator knobs, the wand is connected by a tube to a glass syringe, which is used to generate the pressure that is required to eject the fluid from the needle tip.
To maintain a steady pressure, the syringe is mounted onto a pipe clamp. Using two rubber stoppers and two metal clamps, raise the needle a few millimeters to prevent it from being damaged in later steps and then push back the microscope back. Pillar, increase the pressure by depressing the syringe plunger.
0.5 to two cubic centimeters. Place a Petri dish containing a cover slip with a wounded monolayer. On the viewing stage, pull the microscope back.
Pillar forward to an upright position causing the condenser to be aligned in the needle to enter the liquid. This should be done carefully to prevent the needle from breaking on the cover slip. Using the 10 x objective, identify the center of the cross wound and position the needle above the cells to be injected.
Increase the magnification by switching to the 40 x objective. Then lower and center the needle using the fine adjustment knobs in the joystick. Begin injection at one corner of the wound cross by lowering the needle to make contact with the nucleus.
Once injected, a cell has a notable change in phase brightness. Continue along one edge of the wound cross. For easy identification of injected cells during visualization, it is critical that the injection pressure be properly adjusted for each needle.
The pressure must be high enough to pierce the plasma and nuclear membranes, but not so high as to kill the cells. Micro inject cells along the wound edge for a fixed period of time. With practice, several hundred cells can be injected during this interval.
If injecting plasma DNA incubate the micro injected cells at 37 degrees Celsius in a tissue culture incubator for 20 minutes prior to fixation, then terminate transcription. By treating the cells with one microgram per milliliter alpha, a mantin dissolved in growth media. This concentration effectively inhibits transcription from micro injected plasmids.
Place the cells back in the incubator and allow the cells to export the newly synthesized mRNA. A single needle can be reused to inject multiple cover slips. Although the needle needs to be replaced when the type of DNA or RNA that is micro injected has changed.
Furthermore, once the needle is removed from the wand, it should be disposed of to obtain an accurate measurement of the mRNA nuclear export kinetics. Separate samples should be fixed at zero minutes, 15 minutes, 30 minutes, 60 minutes, and 120 minutes. Post alpha manin treatment at the appropriate time, aspirate the growth media and wash the cells twice in two milliliters of PBS solution.
It is important to keep the cover slips moist by minimizing the time they're not submerged in liquid. Fix the cells by adding two milliliters of 4%Aldehyde in PBS incubate the cells for at least 15 minutes at room temperature. After washing the fixed samples two more times, permeable the cells with two milliliters of 0.1%Triton X 100 in PBS, again, incubate the cells for at least 15 minutes at room temperature.
Finally wash the samples an additional two times to prepare the samples for hybridization. Wash the cells twice with a wash buffer of one XSSC solution containing 25 to 60%form a mid. Then prepare the staining chamber for hybridization by covering the bottom of a 150 millimeter Petri dish with water and floating a piece of paraform on top.
Remove the water by turning the dish over, thus allowing the paraform to adhere to the bottom of the dish. Manually remove any air bubbles for each cover slip to be stained. Pipette 100 microliter drops of the hybridization solution onto the paraform.
Note that the amount of formage should be optimized for the particular fish pro being used with the help of forceps. Remove a cover slip with the fixed cells from the 30 millimeter Petri dish. Using watman filter paper, dry the backside of the cover slip and wick excess buffer from the front side without drying the cells.
Then place the cover slip face down onto the fish solution. Repeat this process for each cover slip. After placing the staining chambers lid back on, incubate the samples for five to 18 hours at 37 degrees Celsius.
Prepare several washing chambers in the same manner as the staining chamber was prepared for each cover slip pipette one milliliter of wash buffer onto the paraform of the washing chamber. Following incubation, remove the cover slips from the staining chamber by pipetting one milliliter of wash buffer. Next to the cover slip, the liquid should be drawn underneath each sample by capillary action.
Then remove the cover slips with forceps and place each onto a drop of wash buffer incubated room temperature for five minutes. Wash each cover slip twice more for a total of three washes for each cover slip pipette 10 to 30 microliters of mounting solution with dappy onto a previously washed slide. Each slide can accommodate two cover slips using forceps and watman filter paper.
Dry the back of the cover slips and wick excess liquid from the front side of the cover slip without drying the samples. Place each cover slip face down onto a drop of mounting solution. Mounted samples can be stored at four degrees Celsius.
Here the effects of alpha mantin on transcription of injected plasma DNA has shown NIH three T three fibroblast cells, which were pretreated with varying concentrations of alpha mantin. Were injected with TFL s delta i, plasmid DNA and OG conjugated 70 kilo and dextran. Each row corresponds to a single field of view imaged for OG 70 kilodalton dextran and T uts delta I mRNA.
It is evident that high concentrations of the drug completely inhibited production of T UTS delta I transcript, whereas this was not observed at lower concentrations.Shown. Here is a time course of mRNA nuclear export cost. Seven cells were injected with T UTS delta i plasma DNA and OG conjugated 70 kilodalton dextran.
30 minutes following injected cells were treated with alpha mantin and incubated at 37 degrees Celsius for zero minutes, 60 minutes and 120 minutes. The cells were then fixed and stained with a probe against T UTS delta I mRNA. And with antibodies against the ER marker trap alpha, each row corresponds to a single field of cells.
For information on how to quantify mRNA export, please refer to the online text here. The 120 minute time point was blown up To demonstrate the colocalization of TFS delta I mRNA in the er, an overlay of the T UTS delta I mRNA in green and the trap alpha in red is shown. Information on how to coast stain mRNA with proteins can be found in the online text.
We've just shown you how to measure the nuclear export kinetics of mRNA by microinjection. When performing this procedure, it's important to remember that a successful microinjection requires proper preparation. Key steps such as DNA and RNA, sample preparation, pulling needles, aligning optical equipment, and troubleshooting microinjection are described in our written protocol.
When imaging and analyzing micro injected mRNA, it is important that quantification is limited to cells that received greater than 90%of the injected fluid into the nucleus. A successful nuclear injection can be assessed by visualizing the distribution of organ green dextran in the nucleus and cytoplasm. Please refer to our online protocol for more information about designing fish probes and for using image analysis software for analyzing the distribution of mRNA in the cytoplasm in the nucleus.
So that's it. Thank you for watching and good luck with your experiments.
