High-throughput di Gene Tagging in

The overall goal of this method is to tag proteins in Trypansoma brucei in a high-throughput manner. This method can help answer key questions in the kinetoplacid field about the localization and potential function of large cohorts of proteins. The main advantage of this technique is that large numbers of proteins can be rapidly and easily tagged in parallel.

Though this method can provide insight into localization, interactions of large cohorts of proteins can also be investigated by using other tags. For example, the BirA tag for a bio-ID proximity mapping experiment. Demonstrating the procedure will be Ross Madden, a research assistant from our laboratory.

Prior to starting this procedure, pre-freeze a 96-well PCR cooler in the minus 80 degrees Celsius freezer. Prepare Master Mix A by combining the following in a 10 milliliter tube. 2, 100 microliters of distilled deionized water, 105 microliters of PCR-grade DMSO, 105 microliters of 10 millimolar dNTPs, and 105 microliters of pPOT template.

Pour Master Mix A into a reagent reservoir and aliquot 23 microliters of Master Mix A into each well of a 96-well PCR plate. Using a P20 12-channel multi-channel pipette, add two microliters of 100 micromolar pooled primers to each loaded well. Seal the plate with film.

Place it onto the pre-chilled PCR cooler and allow it to freeze for a minimum of 20 minutes at minus 80 degrees Celsius. The freezing of PCR Master Mix A is critical to ensure an efficient hot start reaction, even when using a specific hot start polymerase. This leads to a reproducible and high-yield amplification.

While the PCR plate is in the freezer, prepare Master Mix B.Combine the following in a 10 milliliter tube. 2, 048 microliters of distilled deionized water, 525 microliters of 10X buffer, and 52.5 microliters of polymerase, for a total volume of 2, 626 microliters per plate. Remove the PCR plate and PCR cooler from the minus 80 degrees Celsius freezer.

With the plate still on the PCR cooler, quickly add 25 microliters of Master Mix B on top of the frozen Master Mix A in each well for a total reaction volume of 50 microliters. This is time-critical and should be completed before Master Mix A can thaw. Seal the plate with film.

Set the thermal cycler as follows. 94 degrees Celsius for 10 minutes, then, 30 cycles of 94 degrees Celsius for 15 seconds, 65 degrees Celsius for 30 seconds, and 72 degrees Celsius for two minutes, followed by a final extension period of 72 degrees Celsius for seven minutes. After the block has reached 94 degrees Celsius, load the PCR plate in the thermal cycler and start the amplification program.

Begin this procedure by preparing a large 1%agarose gel in Tris-acetate EDTA or TAE running buffer, with four rows of wells. Load a one kb DNA ladder into the first and 28th wells of each row. The next step is to transfer two microliters of PCR product directly, without DNA loading buffer, to each lane of the gel, following the pattern shown here.

For example, the PCR products from Rows A and B of the PCR plate are loaded into the odd-numbered and even-numbered lanes of the first row of the gel, respectively. Working quickly to reduce the possibility of contaminating the amplicons, load the PCR products from Row A of the PCR plate into the odd-numbered lanes of the first row of the gel. Then, load the PCR products from Row B of the PCR plate into the even-numbered lanes of the first row of the gel.

Load the PCR products from Rows C and D of the PCR plate using this same pattern, with Row C into the odd-numbered lanes and Row D into the even-numbered lanes of the second row of the gel. Continue this loading pattern until each well of the PCR plate is loaded onto the gel. Place the gel into the running tank and fill the tank with TAE running buffer until the gel is submerged.

Run at 100 volts for 30 minutes. Visualize using a UV transilluminator. Shown here is a representative 96-well PCR validation agarose gel, where 95 out of 96 PCRs were successful.

The unsuccessful PCR was H11. This procedure uses Trypansoma brucei, procyclic form, cell line SMOXP9. Grow the cells to the appropriate density as described in the protocol text and harvest the required number of cells by centrifugation at 800 times G for 10 minutes.

Begin setting up for the electroporation while the cells are being centrifuged. Connect the plate handler to the electroporator. On the electroporator unit, set the voltage to 1, 500 volts, the pulse length to 100 microseconds, the number of pulses to 12, and the pulse interval to 500 milliseconds.

On the plate handler, set the pulse count to one. Using a P200 12-channel multi-channel pipette, transfer the PCR reactions from the PCR plate to a 96-well disposable electroporation plate. Wash the cells in 10 milliliters of modified cytomix and centrifuge again at 800 times G for 10 minutes.

Resuspend the cells in 23 milliliters of modified cytomix for a final concentration of five times 10 to the seventh cells per milliliter. Transfer the cell suspension to a reagent reservoir. Pipette 200 microliters of the cell solution into each well of the electroporation plate and mix with the PCR product by pipetting.

Use a tissue to remove any droplets from the top of the plate to avoid short circuits. Apply sealing film to the top of the electroporation plate, positioning it so as to leave the holes for the electrodes uncovered at the top and bottom of each column. Avoid covering the raised points used to guide the film.

Load the electroporation plate into the plate handler and close the lid. Press pulse"on the electroporator unit. After electroporation, the cells must be transferred quickly to four 24-well tissue culture plates that contain one milliliter of SDM-79 media per well for recovery.

Use a P200 12-channel multi-channel pipette with every other tip missing, such that the remaining six tips line up with the six rows on a 24-well tissue culture plate. After the cells are transferred from the 96-well plate to the 24-well plate, wash the wells in the 96-well plate with media from the corresponding wells in the 24-well plate. Then, transfer the wash back to the wells in the 24-well plate.

Transfer the electroporated cells from the 96-well electroporation plate to the 24-well tissue culture plates in the pre-defined pattern shown here. For example, cells from the odd-numbered wells of Row A of the 96-well plate are transferred to Row A of Plate A, while cells from the even-numbered wells of Row A from the 96-well plate are transferred to Row A of Plate C.After each transfer of the cells from the 96-well plate to the 24-well plate, wash the wells in the 96-well plate with medium from the corresponding row of the 24-well plate. Then, transfer the wash back to the 24-well plate.

Transferring cells to the 24-well plates is confusing, but you must be able to track the resulting cell lines to the correct gene ID.It is critical to do this step quickly to keep the cells healthy. After all the electroporated cells have been transferred to the four 24-well plates, place the 24-well plates in a clean plastic box with a lid that forms a tight seal. Put the box in a 28 degree Celsius incubator.

Between six to eight hours later, add to each well one milliliter of SDM-79 with 10%FCS containing selective antibiotic. Incubate at 28 degrees Celsius for nine to 10 days until transgenic cell lines become visible. After a 96-well transfection for tagging 96 different proteins on the end terminus, the cells are transferred to 24-well tissue culture plates for recovery and selection.

Each well is then assessed to determine if it contains healthy, drug-resistant cells. A healthy well contains predominantly highly-active cells that are bright in phase-contrast microscopy. In this example schematic, green represents a well with successful transfection, and gray represents a well with only dead cells.

88 out of 96, or 92%of wells, contain successfully-transfected parasites after 15 days of selection. Once mastered, it will take approximately an hour from counting the cells to plating them after electroporation. While attempting this procedure, it's important to remember to add the selective drugs at six to eight hours after transfection.

After watching this video, you should have a good understanding of how to tag large cohorts of proteins in Trypansoma brucei.