SD was supported by a Sir Henry Wellcome Fellowship [092201/Z/10/Z]. This work was funded by Wellcome Trust grants [WT066839MA][104627/Z/14/Z][108445/Z/15/Z] to Professor Keith Gull. We would like to thank Professor Keith Gull for helpful conversations and insights.

1. 96-well Long Primer PCR
<ol>
	<li>Pre-freeze 96-well PCR cooler in -80 &#176;C freezer.</li>
	<li>In a 10 ml tube, prepare Master Mix A: 2,100 &#181;l ddH2O, 105 &#181;l PCR grade DMSO, 105 &#181;l 10 mM dNTPs, 105 &#181;l pPOT template (25 ng/&#181;l) 9, for a total volume of 2,415 &#181;l per plate.</li>
	<li>Aliquot 23 &#181;l into each well of a 96-well PCR plate.</li>
	<li>Add 2 &#181;l of 100 &#181;M pooled primers to each loaded well using a P20 12 channel multichannel pipette. Seal with film, place plate on the pre-chilled PCR cooler and allow to freeze for a minimum of 20 min at -80 &#176;C.</li>
	<li>In a 10 ml tube, prepare Master Mix B: 2,048 &#181;l ddH2O, 525 &#181;l 10x buffer, 52.5 &#181;l polymerase for a total volume of 2,626 &#181;l per plate.</li>
	<li>Remove plate and PCR cooler from the -80 &#176;C freezer.</li>
	<li>With the plate still on the PCR cooler, add 25 &#181;l Master Mix B on top of the frozen Master Mix A for a total reaction volume of 50 &#181;l. This is time critical, and should be completed before Mix A can thaw. Seal plate with film.</li>
	<li>Set thermal cycler as follows: 94 &#176;C for 10 min, then 30 cycles of 94 &#176;C for 15 sec, 65 &#176;C for 30 sec, 72&#176;C for 2 min followed by a final extension period of 72 &#176;C for 7 min.</li>
	<li>Load the PCR plate after the block has reached 94 &#176;C.</li>
</ol>
2. Validation of 96-well Long Primer PCR
<ol>
	<li>Cast a large 1% (w/v) agarose gel in Tris Acetate EDTA (TAE) running buffer with 4 x 28-well combs to give a gel with 4 rows of wells. Align alternate teeth of the comb with the tips of a 12 channel multichannel pipette. Carefully submerge the gel in TAE running buffer after the lanes are loaded.</li>
	<li>Load 1 kb DNA ladder into the 1st and 28th well of each row.</li>
	<li>Using a P20 12 channel multichannel pipette, transfer 2 &#181;l of PCR product directly to each lane of the gel (Figure 1A). Do this quickly to reduce the possibility of contaminating the amplicons.
	<ol>
		<li>Load the PCR products from row A of the PCR plate into the odd numbered lanes of the 1st row of the gel (A1 - lane 3, A2 - lane 5 &#8230;&#8230; A11 - lane 23, A12 - lane 25) and load the PCR products from row B of the PCR plate into the even numbered lanes of the 1st row of the gel (B1 - lane 4, A2 - lane 6 &#8230;&#8230; B11 - lane 24, B12 - lane 26).</li>
		<li>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.</li>
	</ol>
	</li>
	<li>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 &#176;C for several weeks prior to transfection</li>
</ol>
3. 96-well Transfection
<ol>
	<li>Use the T. brucei procyclic form cell line SMOXP9 10 for this procedure and grow in SDM-79 media containing 10% FCS 10.</li>
	<li>Use 1 x 107 cells per transfection (total of 1.1 x 109 cells) when tagging on the N terminus of the protein and 2 x 107 cells per transfection (total of 2.2 x 109 cells) when tagging on the C terminus of the protein. Maintain the cells in mid-log (1.2 x 106-1 x 107 cells/ml) for several days prior to transfection and harvest the cells for transfection at a density of 5-8 x 106 cells/ml.</li>
	<li>Allow frozen PCR products for transfection to thaw at room temperature.</li>
	<li>Count cells using a haemocytometer or automatic cell counter.</li>
	<li>Pellet required number of cells in multiple 50 ml tubes at 800 x g for 10 min.</li>
	<li>After centrifugation discard supernatant and resuspend the cells in 10 ml modified cytomix (0.8 mM EGTA, 24 mM KCl, 0.15 mM CaCl2, 10 mM potassium phosphate buffer pH7.6, 25 mM HEPES-KOH pH7.6, 2.6 mM MgCl2, 0.5% (w/v) glucose, 100 &#181;g/ml BSA, 1 mM hypoxanthine, 144 mM sucrose) per tube. Transfer all cell solutions to a single tube and spin again at 800 x g for 10 min.</li>
	<li>After centrifugation, discard supernatant and resuspend the cells in 23 ml of modified cytomix for a final concentration of 5 x 107 cells/ml.</li>
	<li>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.</li>
	<li>Connect the plate handler to the electroporator. On the electroporator unit set the voltage to 1,500 V, the pulse length to 100 &#181;sec 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.</li>
	<li>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.</li>
	<li>When the cells have been spun and are resuspended to the final concentration of 5 x 107 cells/ml (Step 3.7), transfer to a reagent reservoir. Pipette 200 &#181;l of the cell solution into each well of the electroporation plate using a P200 12 channel multichannel pipette, mix with PCR product by pipetting.</li>
	<li>Using tissue remove any droplets from the top of the plate to avoid short-circuits.</li>
	<li>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.</li>
	<li>Load the electroporation plate into the plate handler and close the lid. Press &#39;Pulse&#39; on the electroporator unit.</li>
	<li>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 24-well 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).</li>
	<li>Prepare the P200 pipette tips - for each 96-well transfection prepare 2 boxes of 96 tips with every other column removed.
	<ol>
		<li>Transfer wells A1/A3/A5/A7/A9/A11 on the 96-well electroporation plate to plate A row A of the 24-well tissue culture plates, B1/B3/B5/B7/B9/B11 into row B, C1/C3/C5/C7/C9/C11 into row C and D1/D3/D5/D7/D9/D11 into row D. After transfer of each set of 6 wells from the 96-well plate to the 24-well plate, the wells in the 96-well plate are then washed with media from the corresponding wells in the 24-well plate and the wash is then transferred back to the wells in the 24-well plate.</li>
		<li>Transfer wells E1/E3/E5/E7/E9/E11 on the 96-well electroporation plate to 24-well plate B row A, F1/F3/F5/F7/F9/F11 into row B, G1/G3/G5/G7/G9/G11 into row C and H1/H3/H5/H7/H9/H11 into row D. After transfer of each set of 6 wells from the 96-well plate to the 24-well plate, the wells in the 96-well plate are then washed with media from the corresponding wells in the 24-well plate and the wash is then transferred back to the wells in the 24-well plate.</li>
		<li>Transfer wells A2/A4/A6/A8/A10/A12 on the 96-well electroporation plate to 24-well plate C row A, B2/B4/B6/B8/B10/B12 into row B, C2/C4/C6/C8/C10/C12 into row C and D2/D4/D6/D8/D10/D12 into row D. After transfer of each set of 6 wells from the 96-well plate to the 24-well plate, the wells in the 96-well plate are then washed with media from the corresponding wells in the 24-well plate and the wash is then transferred back to the wells in the 24-well plate.</li>
		<li>Transfer wells E2/E4/E6/E8/E10/E12 on the 96-well electroporation plate to 24-well plate D row A, F2/F4/F6/F8/F10/F12 into row B, G2/G4/G6/G8/G10/G12 into row C and H2/H4/H6/H8/H10/H12 into row D. After transfer of each set of 6 wells from the 96-well plate to the 24-well plate, the wells in the 96-well plate are then washed with media from the corresponding wells in the 24-well plate and the wash is then transferred back to the wells in the 24-well plate.</li>
	</ol>
	</li>
	<li>Using an inverted phase contrast microscope, examine 1 well from each column to check the cells. There will be a mixture of live and dead cells with a substantial amount of cell debris. Live cells can be distinguished from dead cells because they will be bright under phase contrast microscopy and will be moving.</li>
	<li>Place the 24-well plates in a clean, plastic box with a lid that forms a tight seal. Put the box in a 28 &#176;C incubator.</li>
	<li>Between 6-8 hr later, add 1 ml of SDM-79 with 10% FCS containing 2x selective antibiotic to each well. In our experience, cell lines where the genes are tagged on the N terminus are resistant to higher concentrations of selective drug. Therefore, using blasticidin as an example, tagging on the N terminus requires a final concentration of 20 &#181;g/ml blasticidin and tagging on the C terminus requires a final concentration of 10 &#181;g/ml blasticidin. 
	Note: Transgenic cell lines will become visible 9-10 days after transfection.</li>
	<li>Passage at least twice in fresh drug and media prior to analysis. Figure 2B shows the results from a typical 96-well transfection for tagging 96 different proteins on the N terminus. Assess each well by phase contrast microscopy to determine if it contains healthy, drug resistant cells. A healthy well contains predominantly (&#60;95%) highly active cells that are bright in phase contract microscopy. Subsequent analysis by epifluorescence microscopy or western blot is required to determine if the protein has been successfully tagged.</li>
</ol>

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	<li>Kelly, S., Reed, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+genomics+in+Trypanosoma+brucei:+a+collection+of+vectors+for+the+expression+of+tagged+proteins+from+endogenous+and+ectopic+gene+loci.">Functional genomics in Trypanosoma brucei: a collection of vectors for the expression of tagged proteins from endogenous and ectopic gene loci.</a> Mol Biochem Parasitol. 154 (1), 103-109 (2007).</li><li>Wirtz, L. E., Leal, S., Ochatt, C., Cross, G. A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+tightly+regulated+inducible+expression+system+for+conditional+gene+knock-outs+and+dominant-negative+genetics+in+Trypanosoma+brucei.">A tightly regulated inducible expression system for conditional gene knock-outs and dominant-negative genetics in Trypanosoma brucei.</a> Mol Biochem Parasitol. 99 (1), 89-101 (1999).</li><li>Shi, H., Djikeng, A., Mark, T., Wirtz, L. E., Tschudi, C., Ullu, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+interference+in+Trypanosoma+brucei+by+heritable+and+inducible+double-stranded+RNA.">Genetic interference in Trypanosoma brucei by heritable and inducible double-stranded RNA.</a> RNA. 6 (7), 1069-1076 (2000).</li><li>Alsford, S., Turner, D. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+phenotyping+using+parallel+sequencing+of+RNA+interference+targets+in+the+African+trypanosome.">High-throughput phenotyping using parallel sequencing of RNA interference targets in the African trypanosome.</a> Genome Res. 21 (6), 915-924 (2011).</li><li>Alsford, S., Eckert, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+decoding+of+antitrypanosomal+drug+efficacy+and+resistance.">High-throughput decoding of antitrypanosomal drug efficacy and resistance.</a> Nature. 482 (7384), 232-236 (2012).</li><li>Mony, B. M., MacGregor, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+dissection+of+the+quorum+sensing+signalling+pathway+in+Trypanosoma+brucei.">Genome-wide dissection of the quorum sensing signalling pathway in Trypanosoma brucei.</a> Nature. 505 (7485), 681-685 (2014).</li><li>Rigaut, G., Shevchenko, A., Rutz, B., Wilm, M., Mann, M., S&#233;raphin, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+generic+protein+purification+method+for+protein+complex+characterization+and+proteome+exploration+:+Abstract+Nature+Biotechnology.">A generic protein purification method for protein complex characterization and proteome exploration : Abstract Nature Biotechnology.</a> Nature Biotechnol. 17 (10), 1030-1032 (1999).</li><li>Los, G. V., Encell, L. 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The authors have nothing to disclose.

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 forms&#160;&#8211; other 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/

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 1-3. Advances in proteomics and sequencing have resulted in large datasets that highlight potentially interesting genes 4-6; however, many genes have minimal information associated with them in the existing databases. There is therefore a need for a high-throughput method to aid protein functional characterization.

Expression of a tagged protein can give a multiplicity of insights into a protein&#39;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 7, HaloTag 8 or His tag can be purified for biochemical assays and identification of its interaction partners.

We recently developed a robust tagging methodology for T. brucei&#160; 9. This used long primer PCR to generate the DNA for transfection and allowed the tagging of dozens of proteins in parallel&#160;&#8211; a major improvement to existing protocols. We have now improved the scalability of this protocol in procyclic forms by an order of magnitude. Here, we present our method where we perform the PCR and transfection into 96-well plates, with the recovery and selection occurring in 24-well plates. As hundreds of proteins can now be tagged in parallel this method provides a cost-effective and feasible method for tagging the entire trypanosome genome.

<table border="0" cellpadding="0" cellspacing="0" width="617">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Oligonucleotide</td>
			<td style="width:115px;">Life technologies</td>
			<td style="width:119px;">na</td>
			<td style="width:167px;">desalt only (do not use additional purification)</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">DMSO</td>
			<td style="width:115px;">Roche</td>
			<td style="width:119px;">Included with the Expand HiFi pack</td>
			<td>Must be PCR grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">dNTP</td>
			<td style="width:115px;">any reputable</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Expand HiFi polymerase</td>
			<td style="width:115px;">Roche</td>
			<td style="width:119px;">11759078001</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">BTX ECM830</td>
			<td style="width:115px;">Harvard Apparatus</td>
			<td style="width:119px;"></td>
			<td>Electroporator</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">HT-200 plate handler</td>
			<td style="width:115px;">Harvard Apparatus</td>
			<td style="width:119px;">HT-200</td>
			<td>Handles the 96-well eletroplates</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">MOS 96 ELECTROPLATE 4mm</td>
			<td style="width:115px;">Harvard Apparatus</td>
			<td style="width:119px;">45-0452</td>
			<td>Electroplate for the 96-well electroporation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Blasticidin S Hydrochloride</td>
			<td>Melford</td>
			<td style="width:119px;">3513-03-9</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hygromycin b Gold</td>
			<td>Invivogen</td>
			<td style="width:119px;">ant-hg-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Phleomycin</td>
			<td style="width:115px;">Melford</td>
			<td style="width:119px;">P0187</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">225cm TC Flask, canted neck, phenolic cap</td>
			<td style="width:115px;">Appletonwoods</td>
			<td style="width:119px;">BC006</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">24 well culture plates</td>
			<td style="width:115px;">Appletonwoods</td>
			<td style="width:119px;">BC017</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Eppendorf&#174; PCR Cooler, iceless cold storage system for 96 well plates and PCR tubes</td>
			<td style="width:115px;">Sigma Aldrich</td>
			<td style="width:119px;">Z606634-1EA</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">96-well Multiply&#174; PCR plate with lateral skirt</td>
			<td style="width:115px;">Sarstedt</td>
			<td style="width:119px;">72.1979.203</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

In this representative transfection, the primers were designed using the TagIt perl script 9 and synthesized commercially. The 96-well PCR was performed and validated as described (Figure 1A); in this example, 95/96 PCRs were successful (Figure 1B). In our experience, repeating failed reactions either with the original primers or re-synthesized primers does not result in a successful PCR.
The amplicons were transferred into 96-well electroporation plates for electroporation (Figure 2A) and then transferred to 24-well culture plates for recovery and selection. After sufficient time to allow the selection to occur, the wells were scored for survival prior to further analysis. In this example, 88/96 wells (92%) were positive after 15 days selection.
<img alt="Figure 1" src="/files/ftp_upload/54342/54342fig1.jpg" /> 
Figure 1: Validation of long primer PCR. (A) Pattern for transferring PCR products from 96-well PCR plate to the agarose gel for validation of PCR. (B) A representative 96-well PCR validation gel for tagging 95 genes on the N terminus, and 1 amplicon containing no 5&#39; targeting homology to act as a negative control in the transfection. The samples are loaded onto a 1% (w/v) agarose gel and resolved at 100 V for 30 min. <a href="https://www.jove.com/files/ftp_upload/54342/54342fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54342/54342fig2.jpg" /> 
Figure 2: 96-well electroporation and selection. (A) Pattern for transferring electroporated cells from 96-well electroporation plate to 24-well tissue culture plates. (B) A schematic showing a typical live/dead scoring after 15 days of selection in 24-well plates. Green represents a successful transfection, grey represents a well with only dead cells. In this example, 88/96 (92%) of wells contained successfully transfected parasites. <a href="https://www.jove.com/files/ftp_upload/54342/54342fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about High-throughput Gene Tagging in Trypanosoma brucei at JoVE.com

High-throughput Gene Tagging in Trypanosoma brucei

Addition of a tag to a protein is a powerful way of gaining insight into its function. Here, we describe a protocol to endogenously tag hundreds of Trypanosoma brucei proteins in parallel such that genome scale tagging is achievable.

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 ...

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Research

JoVE Journal

Immunology and Infection

7.9K Views.  University of Oxford. Addition of a tag to a protein is a powerful way of gaining insight into its function. Here, we describe a protocol to endogenously tag hundreds of Trypanosoma brucei proteins in parallel such that genome scale tagging is achievable.

Video: High-throughput Gene Tagging in Trypanosoma brucei

Examination of the Telomere G-overhang Structure in Trypanosoma brucei

The telomere G-overhang structure has been identified in many eukaryotes including yeast, vertebrates, and Trypanosoma brucei. It serves as the substrate for telomerase for de novo telomere DNA synthesis and is therefore important for telomere maintenance. T. brucei is a protozoan parasite that causes sleeping sickness in humans and nagana in cattle. Once infected mammalian host, T. brucei cell regularly switches its surface antigen to evade the host's immune attack. We have recently demonstrated that the T. brucei telomere structure plays an essential role in regulation of surface antigen gene expression, which is critical for T. brucei pathogenesis. However, T. brucei telomere structure has not been extensively studied due to the limitation of methods for analysis of this specialized structure. We have now successfully adopted the native in-gel hybridization and ligation-mediated primer extension methods for examination of the telomere G-overhang structure and an adaptor ligation method for determination of the telomere terminal nucleotide in T. brucei cells. Here, we will describe the protocols in detail and compare their different advantages and limitations.

Examination of the Telomere G-overhang Structure in Trypanosoma brucei

Telomeres are essential for chromosome stability and the telomere G-overhang structure is essential for telomerase-mediated telomere maintenance. We have recently adopted two methods for detecting the telomere G-overhang structure in Trypanosoma brucei, which are native in-gel hybridization and ligation-mediated primer extension, which will be described.

The telomere G-overhang structure has been identified in many eukaryotes including yeast, vertebrates, and Trypanosoma brucei. It serves as the substrate ...

High-throughput Detection Method for Influenza Virus

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, precise diagnosis of the infecting strain and rapid high-throughput screening of vast numbers of clinical samples is paramount to control the spread of pandemic infections. Current clinical diagnoses of influenza infections are based on serologic testing, polymerase chain reaction, direct specimen immunofluorescence and cell culture 1,2.
Here, we report the development of a novel diagnostic technique used to detect live influenza viruses. We used the mouse-adapted human A/PR/8/34 (PR8, H1N1) virus 3 to test the efficacy of this technique using MDCK cells 4. MDCK cells (104 or 5 x 103 per well) were cultured in 96- or 384-well plates, infected with PR8 and viral proteins were detected using anti-M2 followed by an IR dye-conjugated secondary antibody. M2 5 and hemagglutinin 1 are two major marker proteins used in many different diagnostic assays. Employing IR-dye-conjugated secondary antibodies minimized the autofluorescence associated with other fluorescent dyes. The use of anti-M2 antibody allowed us to use the antigen-specific fluorescence intensity as a direct metric of viral quantity. To enumerate the fluorescence intensity, we used the LI-COR Odyssey-based IR scanner. This system uses two channel laser-based IR detections to identify fluorophores and differentiate them from background noise. The first channel excites at 680 nm and emits at 700 nm to help quantify the background. The second channel detects fluorophores that excite at 780 nm and emit at 800 nm. Scanning of PR8-infected MDCK cells in the IR scanner indicated a viral titer-dependent bright fluorescence. A positive correlation of fluorescence intensity to virus titer starting from 102-105 PFU could be consistently observed. Minimal but detectable positivity consistently seen with 102-103 PFU PR8 viral titers demonstrated the high sensitivity of the near-IR dyes. The signal-to-noise ratio was determined by comparing the mock-infected or isotype antibody-treated MDCK cells.
Using the fluorescence intensities from 96- or 384-well plate formats, we constructed standard titration curves. In these calculations, the first variable is the viral titer while the second variable is the fluorescence intensity. Therefore, we used the exponential distribution to generate a curve-fit to determine the polynomial relationship between the viral titers and fluorescence intensities. Collectively, we conclude that IR dye-based protein detection system can help diagnose infecting viral strains and precisely enumerate the titer of the infecting pathogens.

This method describes the use of Infrared dye based imaging system for detection of H1N1 in bronchioalveolar lavage (BAL) fluid of infected mice at a high sensitivity. This methodology can be performed in a 96- or 384-well plate, requires &lt;10 &mu;l volume of test material and has the potential for concurrent screening of multiple pathogens.

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, ...

Quantitative High-throughput Single-cell Cytotoxicity Assay For T Cells

Cancer immunotherapy can harness the specificity of immune response to target and eliminate tumors. Adoptive cell therapy (ACT) based on the adoptive transfer of T cells genetically modified to express a chimeric antigen receptor (CAR) has shown considerable promise in clinical trials1-4. There are several advantages to using CAR+ T cells for the treatment of cancers including the ability to target non-MHC restricted antigens and to functionalize the T cells for optimal survival, homing and persistence within the host; and finally to induce apoptosis of CAR+ T cells in the event of host toxicity5.
Delineating the optimal functions of CAR+ T cells associated with clinical benefit is essential for designing the next generation of clinical trials. Recent advances in live animal imaging like multiphoton microscopy have revolutionized the study of immune cell function in vivo6,7. While these studies have advanced our understanding of T-cell functions in vivo, T-cell based ACT in clinical trials requires the need to link molecular and functional features of T-cell preparations pre-infusion with clinical efficacy post-infusion, by utilizing in vitro assays monitoring T-cell functions like, cytotoxicity and cytokine secretion. Standard flow-cytometry based assays have been developed that determine the overall functioning of populations of T cells at the single-cell level but these are not suitable for monitoring conjugate formation and lifetimes or the ability of the same cell to kill multiple targets8.
Microfabricated arrays designed in biocompatible polymers like polydimethylsiloxane (PDMS) are a particularly attractive method to spatially confine effectors and targets in small volumes9. In combination with automated time-lapse fluorescence microscopy, thousands of effector-target interactions can be monitored simultaneously by imaging individual wells of a nanowell array. We present here a high-throughput methodology for monitoring T-cell mediated cytotoxicity at the single-cell level that can be broadly applied to studying the cytolytic functionality of T cells.

We describe a single-cell high-throughput assay to measure cytotoxicity of T cells when incubated with tumor target cells. This method employs a dense, elastomeric array of sub-nanoliter wells (~100,000 wells/array) to spatially confine the T cells and target cells at defined ratios and is coupled to fluorescence microscopy to monitor effector-target conjugation and subsequent apoptosis.

Cancer immunotherapy can harness the specificity of  immune response to target and eliminate tumors. Adoptive cell therapy (ACT)  based on the adoptive ...

Scalable High Throughput Selection From  Phage-displayed Synthetic Antibody Libraries

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. However, it is apparent that traditional monoclonal technologies are not alone up to this task. This has led to the development of alternate methods to satisfy the demand for high quality and renewable affinity reagents to all accessible elements of the proteome. Toward this end, high throughput methods for conducting selections from phage-displayed synthetic antibody libraries have been devised for applications involving diverse antigens and optimized for rapid throughput and success. Herein, a protocol is described in detail that illustrates with video demonstration the parallel selection of Fab-phage clones from high diversity libraries against hundreds of targets using either a manual 96 channel liquid handler or automated robotics system. Using this protocol, a single user can generate hundreds of antigens, select antibodies to them in parallel and validate antibody binding within 6-8 weeks. Highlighted are: i) a viable antigen format, ii) pre-selection antigen characterization, iii) critical steps that influence the selection of specific and high affinity clones, and iv) ways of monitoring selection effectiveness and early stage antibody clone characterization. With this approach, we have obtained synthetic antibody fragments (Fabs) to many target classes including single-pass membrane receptors, secreted protein hormones, and multi-domain intracellular proteins. These fragments are readily converted to full-length antibodies and have been validated to exhibit high affinity and specificity. Further, they have been demonstrated to be functional in a variety of standard immunoassays including Western blotting, ELISA, cellular immunofluorescence, immunoprecipitation and related assays. This methodology will accelerate antibody discovery and ultimately bring us closer to realizing the goal of generating renewable, high quality antibodies to the proteome.

A method is described with visual accompaniment for conducting scalable, high throughput selections from phage-displayed combinatorial synthetic antibody libraries against hundreds of antigens simultaneously. Using this parallel approach, we have isolated antibody fragments that exhibit high affinity and specificity for diverse antigens that are functional in standard immunoassays.

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. ...

Detection of Trypanosoma brucei Variant Surface Glycoprotein Switching by Magnetic Activated Cell Sorting and Flow Cytometry

Trypanosoma brucei, a protozoan parasite that causes both Human and Animal African Trypanosomiasis (known as sleeping sickness and nagana, respectively) cycles between a tsetse vector and a mammalian host. It evades the mammalian host immune system by periodically switching the dense, variant surface glycoprotein (VSG) that covers its surface. The detection of antigenic variation in Trypanosoma brucei can be both cumbersome and labor intensive. Here, we present a method for quantifying the number of parasites that have 'switched' to express a new VSG in a given population. The parasites are first stained with an antibody against the starting VSG, and then stained with a secondary antibody attached to a magnetic bead. Parasites expressing the starting VSG are then separated from the rest of the population by running the parasites over a column attached to a magnet. Parasites expressing the dominant, starting VSG are retained on the column, while the flow-through contains parasites that express a new VSG as well as some contaminants expressing the starting VSG. This flow-through population is stained again with a fluorescently labeled antibody against the starting VSG to label contaminants, and propidium iodide (PI), which labels dead cells. A known number of absolute counting beads that are visible by flow cytometry are added to the flow-through population. The ratio of beads to number of cells collected can then be used to extrapolate the number of cells in the entire sample. Flow cytometry is used to quantify the population of switchers by counting the number of PI negative cells that do not stain positively for the starting, dominant VSG. The proportion of switchers in the population can then be calculated using the flow cytometry data.

Detection of Trypanosoma brucei Variant Surface Glycoprotein Switching by Magnetic Activated Cell Sorting and Flow Cytometry

African trypanosomes grown in vitro undergo antigenic variation at a low rate, such that populations are made up of parasites expressing a dominant variant surface glycoprotein (VSG) type and a small population of "switched" variants. This protocol describes a fast method for detecting and quantifying these populations.

Trypanosoma brucei, a protozoan parasite that causes both Human and Animal African Trypanosomiasis (known as sleeping sickness and nagana, respectively) ...

High-throughput Detection of Respiratory Pathogens in Animal Specimens by Nanoscale PCR

Nanoliter scale real-time PCR uses spatial multiplexing to allow multiple assays to be run in parallel on a single plate without the typical drawbacks of combining reactions together. We designed and evaluated a panel based on this principle to rapidly identify the presence of common disease agents in dogs and horses with acute respiratory illness. This manuscript describes a nanoscale diagnostic PCR workflow for sample preparation, amplification, and analysis of target pathogen sequences, focusing on procedures that are different from microliter scale reactions. In the respiratory panel presented, 18 assays were each set up in triplicate, accommodating up to 48 samples per plate. A universal extraction and pre-amplification workflow was optimized for high-throughput sample preparation to accommodate multiple matrices and DNA and RNA based pathogens. Representative data are presented for one RNA target (influenza A matrix) and one DNA target (equine herpesvirus 1). The ability to quickly and accurately test for a comprehensive, syndrome-based group of pathogens is a valuable tool for improving efficiency and ergonomics of diagnostic testing and for acute respiratory disease diagnosis and management.

High-throughput testing of DNA and RNA based pathogens by nanoscale PCR is described using a syndromic canine and equine respiratory PCR panel.

Nanoliter scale real-time PCR uses spatial multiplexing to allow multiple assays to be run in parallel on a single plate without the typical drawbacks of ...

Building Up a High-throughput Screening Platform to Assess the Heterogeneity of HER2 Gene Amplification in Breast Cancers

Targeted therapies against the human epidermal growth factor receptor 2 (HER2) have radically changed the outcome of patients with HER2-positive breast cancers. However, a minority of cases displays a heterogeneous distribution of HER2-positive cells, which generates major clinical challenges. To date, no reliable and standardized protocols for the characterization and quantification of HER2 heterogeneous gene amplification in large cohorts have been proposed. Here, we present a high-throughput methodology to simultaneously assess the HER2 status across different topographic areas of multiple breast cancers. In particular, we illustrate the laboratory procedure to construct enhanced tissue microarrays (TMAs) incorporating a targeted mapping of the tumors. All TMA parameters have been specifically optimized for the silver in situ hybridization (SISH) of formalin-fixed paraffin-embedded (FFPE) breast tissues. Immunohistochemical analysis of the prognostic and predictive biomarkers (i.e., ER, PR, Ki67, and HER2) should be performed using automated procedures. A customized SISH protocol has been implemented to allow a high-quality molecular analysis across multiple tissues that underwent different fixation, processing, and storage procedures. In this study, we provide a proof-of-principle that specific DNA sequences could be localized simultaneously in distinct topographic areas of multiple and heterogeneously processed breast cancers using an efficient and cost-effective method.

Heterogeneous distribution of HER2-positive cells can be observed in a subset of breast cancers and generates clinical dilemmas. Here, we introduce a reliable and cost-effective protocol to define, quantify, and compare HER2 intra-tumor genetic heterogeneity in a large series of heterogeneously processed breast cancers.

Targeted therapies against the human epidermal growth factor receptor 2 (HER2) have radically changed the outcome of patients with HER2-positive breast ...

A High-throughput, High-content, Liquid-based C. elegans Pathosystem

The number of new drugs identified by traditional, in vitro screens has waned, reducing the success of this approach in the search for new weapons to combat multiple drug resistance. This has led to the conclusion that researchers do not only need to find new drugs, but also need to develop new ways of finding them. Amongst the most promising candidate methods are whole-organism, in vivo assays that use high-throughput, phenotypic readouts and hosts that range from Caenorhabditis elegans to Danio rerio. These hosts have several powerful advantages, including dramatic reductions in false positive hits, as compounds that are toxic to the host and/or biounavailable are typically dropped in the initial screen, prior to costly follow up.
Here we show how our assay has been used to interrogate host variation in the well-documented C. elegans&#8212;Pseudomonas aeruginosa liquid killing pathosystem. We also demonstrate several extensions of this well-worked out technique. For example, we are able to carry out high-throughput genetic screens using RNAi in 24- or 96-well plate formats to query host factors in this host-pathogen interaction. Using this assay, whole genome screens can be completed in only a few months, which can dramatically simplify the task of identifying drug targets, potentially without the need for laborious biochemical purification approaches.
We also report here a variation of our method that substitutes the gram-positive bacterium Enterococcus faecalis for the gram-negative pathogen P. aeruginosa. Much as is the case for P. aeruginosa, killing by E. faecalis is time-dependent. Unlike previous C. elegans&#8212;E. faecalis assays, our assay for E. faecalis does not require preinfection, improving its safety profile and reducing the chances of contaminating liquid-handling equipment. The assay is highly robust, showing ~95% death rates 96 h post infection.

A High-throughput, High-content, Liquid-based C. elegans Pathosystem

Here we describe a protocol that is an adaptable, whole host, high-content screening tool that can be utilized to study host-pathogen interactions and be used for drug discovery.

The number of new drugs identified by traditional, in vitro screens has waned, reducing the success of this approach in the search for new weapons to ...

High-throughput Measurement of Plasma Membrane Resealing Efficiency in Mammalian Cells

In their physiological environment, mammalian cells are often subjected to mechanical and biochemical stresses that result in plasma membrane damage. In response to these damages, complex molecular machineries rapidly reseal the plasma membrane to restore its barrier function and maintain cell survival. Despite 60 years of research in this field, we still lack a thorough understanding of the cell resealing machinery. With the goal of identifying cellular components that control plasma membrane resealing or drugs that can improve resealing, we have developed a fluorescence-based high-throughput assay that measures the plasma membrane resealing efficiency in mammalian cells cultured in microplates. As a model system for plasma membrane damage, cells are exposed to the bacterial pore-forming toxin listeriolysin O (LLO), which forms large 30-50 nm diameter proteinaceous pores in cholesterol-containing membranes. The use of a temperature-controlled multi-mode microplate reader allows for rapid and sensitive spectrofluorometric measurements in combination with brightfield and fluorescence microscopy imaging of living cells. Kinetic analysis of the fluorescence intensity emitted by a membrane impermeant nucleic acid-binding fluorochrome reflects the extent of membrane wounding and resealing at the cell population level, allowing for the calculation of the cell resealing efficiency. Fluorescence microscopy imaging allows for the enumeration of cells, which constitutively express a fluorescent chimera of the nuclear protein histone 2B, in each well of the microplate to account for potential variations in their number and allows for eventual identification of distinct cell populations. This high-throughput assay is a powerful tool expected to expand our understanding of membrane repair mechanisms via screening for host genes or exogenously added compounds that control plasma membrane resealing.

Here we describe a high-throughput fluorescence-based assay that measures the plasma membrane resealing efficiency through fluorometric and imaging analyses in living cells. This assay can be used for screening drugs or target genes that regulate plasma membrane resealing in mammalian cells.

In their physiological environment, mammalian cells are often subjected to mechanical and biochemical stresses that result in plasma membrane damage. In ...

High Throughput In Vitro Assessment of Latency Reversing Agents on HIV Transcription and Splicing

HIV remains incurable due to the existence of a reservoir of cells that harbors stable and latent form of the virus, which stays invisible to the immune system and is not targeted by the current antiretroviral therapy (cART). Transcription and splicing have been shown to reinforce HIV-1 latency in resting CD4+ T cells. Reversal of latency by the use of latency reversal agents (LRAs) in the &#34;shock and kill&#34; approach has been studied extensively in an attempt to purge this reservoir but has thus far not shown any success in clinical trials due to the lack of development of adequate small molecules that can efficiently perturb this reservoir. The protocol presented here provides a method for reliably and efficiently assessing latency reversal agents (LRAs) on HIV transcription and splicing. This approach is based on the use of an LTR-driven dual color reporter that can simultaneously measure the effect of an LRA on transcription and splicing by flow cytometry. The protocol described here is adequate for adherent cells as well as the cells in suspension. It is useful for testing a large number of drugs in a high throughput system. The method is technically simple to implement and cost-effective. In addition, the use of flow cytometry allows the assessment of cell viability and thus drug toxicity at the same time.

A high throughput protocol for functional assessment of HIV efficient reactivation and clearance of latent proviruses is described and applied by testing the impact of interventions on HIV transcription and splicing. Representative results of the effect of latency reversing agents on LTR-driven transcription and splicing are provided.

HIV remains incurable due to the existence of a reservoir of cells that harbors stable and latent form of the virus, which stays invisible to the immune ...