High-throughput Gene Tagging in Trypanosoma brucei

Improvements in mass spectrometry, sequencing and bioinformatics have generated large datasets of potentially interesting genes. Tagging these proteins can give insights into their function by determining their localization within the cell and enabling interaction partner identification. We recently published a fast and scalable method to generate Trypanosoma brucei cell lines that express a tagged protein from the endogenous locus. The method was based on a plasmid we generated that, when coupled with long primer PCR, can be used to modify a gene to encode a protein tagged at either terminus. This allows the tagging of dozens of trypanosome proteins in parallel, facilitating the large-scale validation of candidate genes of interest. This system can be used to tag proteins for localization (using a fluorescent protein, epitope tag or electron microscopy tag) or biochemistry (using tags for purification, such as the TAP (tandem affinity purification) tag). Here, we describe a protocol to perform the long primer PCR and the electroporation in 96-well plates, with the recovery and selection of transgenic trypanosomes occurring in 24-well plates. With this workflow, hundreds of proteins can be tagged in parallel; this is an order of magnitude improvement to our previous protocol and genome scale tagging is now possible.


Introduction
Trypanosoma brucei is a protozoan parasite that causes human African trypanosomasis and nagana in cattle. T. brucei is an ideal organism for the analysis of protein function due to the combination of a high quality genome, numerous proteomics and transcriptomics datasets and well developed molecular tools [1][2][3] . Advances in proteomics and sequencing have resulted in large datasets that highlight potentially interesting genes [4][5][6] ; however, many genes have minimal information associated with them in the existing databases. There is therefore a need for a highthroughput method to aid protein functional characterization.
Expression of a tagged protein can give a multiplicity of insights into a protein's function. For example, a protein tagged with a fluorescent protein or epitope can be localized by fluorescence microscopy, which gives information about where the protein might be exerting its biological effect. Alternatively, a protein tagged with a TAP 3. Using a P20 12 channel multichannel pipette, transfer 2 µl of PCR product directly to each lane of the gel ( Figure 1A). Do this quickly to reduce the possibility of contaminating the amplicons. Load the PCR products from row C and D of the PCR plate using the same pattern as described above, with row C into the odd numbered lanes and row D into the even numbered lanes. Continue this loading pattern until each well of the PCR plate is loaded onto the gel. DNA loading buffer is not required to load this gel.
4. Place the gel into the running tank and fill the tank with TAE running buffer until the gel is submerged. Run at 100 V for 30 min and visualize using a UV transilluminator. See Figure 1B for an example gel. Note: PCR products can be stored at -20 °C for several weeks prior to transfection 8. While cells are spinning, add 1 ml of SDM-79 media to each well of 4 x 24-well tissue culture plates. Label the plates A-D and draw a ring around well A1 on each plate in permanent marker to help plate orientation. 9. Connect the plate handler to the electroporator. On the electroporator unit set the voltage to 1,500 V, the pulse length to 100 µsec and the number of pulses to 12 and the pulse interval to 500 msec. On the plate handler, set the pulse count to 1. This ensures that that electroporator will apply a single pulse to each of the 12 columns of the electroporation plate. 10. Using a P200 12 channel multichannel pipette, transfer the PCR reactions from the PCR plate to the 96-well disposable electroporation plates, 4 mm gap. 11. When the cells have been spun and are resuspended to the final concentration of 5 x 10 7 cells/ml (Step 3.7), transfer to a reagent reservoir.

96-well Transfection
Pipette 200 µl of the cell solution into each well of the electroporation plate using a P200 12 channel multichannel pipette, mix with PCR product by pipetting. 12. Using tissue remove any droplets from the top of the plate to avoid short-circuits. 13. Apply the sealing film provided in the plate packaging 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. 14. Load the electroporation plate into the plate handler and close the lid. Press 'Pulse' on the electroporator unit. 15. After electroporation, quickly transfer the cells from the 96-well electroporation plate to the 4 x 24-well tissue culture plates. To transfer the cells, use a P200 12 channel multichannel pipette with every other tip missing such that the remaining 6 tips line up with the 6 rows on a 24well tissue culture plate. Transfer the electroporated cells in the 96-well electroporations plates in pre-defined pattern to the 24-well tissue culture plates (Figure 2A). 16. Prepare the P200 pipette tips -for each 96-well transfection prepare 2 boxes of 96 tips with every other column removed.

Discussion
Dramatic improvements in the sensitivity of proteomics and transcriptomics methods in the last 5-10 years has provided valuable data on thousands of genes and their products. However, the tools to address the function of these proteins have not kept pace.
Tagging a protein facilitates numerous experiments to determine its function. For example, a protein can be fused to a fluorescent protein in a variety of different colors to facilitate localization and co-localization studies. Tags developed for electron microscopy, such as APEX2 or miniSOG 11,12 , allow ultrastructural localization of the tagged protein. Tags for biochemistry, such as the TAP tag, and ProtC-TEV-ProtA (PTP) tag 7,13 allow purification of complexes associated with the protein for identification of binding partners or in vitro biochemical assays.
The specific steps that are critical to the success of the protocol are: the incorporation of DMSO into the PCR Master Mix 1, freezing of the PCR Master Mix 1 prior to the addition of the Master Mix 2, the use of the commercial polymerase in Master Mix 2 and the modification of the cytomix electroporation buffer. In our experience, it is necessary to use double the number of cells for C terminal tagging transfections as for N terminal tagging transfections in order to achieve a similar proportion of positive wells. Therefore, all steps should be performed as described.
This technique is only likely to be successful when transfecting the insect procyclic form trypanosome. Bloodstream forms have a lower transfection efficiency 14 , moreover they are likely to die during selection due to density-dependent toxicity that is unrelated to the selective drug. Therefore, our previous protocol represents the current best technology for tagging of bloodstream form trypanosomes 9 . It is also likely that the transfection efficiency will vary dependent upon the specific trypanosome isolate. This protocol was optimized using 927 SMOX procyclic formsother strains may require additional optimization. Measures that may increase the probability of success include: increasing the amount of PCR amplicon, increasing the number of cells included in the transfection.
We present a method where hundreds of proteins can be tagged in parallel. This will facilitate large scale studies on localization and interaction, complementing existing large datasets and providing invaluable information to the community. Primer templates are available upon request and a list of plasmids templates is available from: http://www.sdeanresearch.com/

Disclosures
The authors have nothing to disclose.