We thank Timothy J. Kurtti and Benjamin Cull for their insightful discussions and suggestions. This study was financially supported by a grant to U.G.M. from the NIH (2R01AI049424) and a grant to U.G.M. from the Minnesota Agricultural Experiment Station (MIN-17-078).

1. Propagation and purification of R. parkeri from tick cell culture
NOTE: All cell culture procedures are to be performed in a class II biosafety cabinet.
<ol>
	<li>Preparing R. parkeri-infected tick cells
	<ol>
		<li>Grow ISE6 cells in 25 cm2 cell culture flasks at 34 &#176;C in 5 mL of L15C300 medium17, supplemented with 5% fetal bovine serum (FBS), 5% tryptose phosphate broth (TPB), and 0.1% lipoprotein concentrate (LPC). 
		NOTE: ISE6 (Ixodes scapularis embryo-derived cell line) is a tick cell line extensively used in many laboratories and is, thus, an essential model to study tick-pathogen interactions17,18,19. The growth rate of ISE6 cells is slower than that of mammalian cells, with a population doubling time of &#8805;72 h. For example, ISE6 cells subcultivated from a 100% confluent culture at a ratio of 1:5 will take 3-4 weeks to regain 100% confluency. Healthy ISE6 cells will be generally rounded with numerous adhering filaments17.</li>
		<li>Count the ISE6 cells with a hemocytometer under a light microscope. Use at a concentration of ~106 cells/mL (100% confluent).
		<ol>
			<li>Gently rinse the cells from the flask with medium and disperse the cells by pipetting to generate a homogenous cell suspension. Add 20 &#181;L of cell suspension between the hemocytometer and cover glass to count the cells using a hemocytometer.</li>
		</ol>
		</li>
		<li>Add host cell-free wild-type R. parkeri20&#160;(average number: 5 &#215; 107-10 &#215; 107) to the ISE6 cell cultures in 25 cm2 cell culture flasks. Incubate infected R. parkeri cell cultures at 34 &#176;C in 5 mL of complete medium consisting of L15C300 medium, 10% FBS, 5% TPB, 0.1% LPC, 0.25% sodium bicarbonate (NaHCO3), and 25 mM HEPES until 90%-100% of the cells are infected. 
		NOTE: The infection rate of R. parkeri in ISE6 cells is determined by Giemsa staining, and the incubation time to reach 90%-100% infection ranges from 5 days to 7 days on average.</li>
		<li>Determine the infection rate via Giemsa staining.
		<ol>
			<li>In the biosafety cabinet, resuspend the infected cell cultures and transfer 50 &#181;L of the suspension to a 1.5 mL microcentrifuge tube.</li>
			<li>Dilute the suspension with complete medium (1:5 dilution) and centrifuge 100 &#181;L onto glass microscope slides using a centrifuge at 113 &#215; g for 5 min. To keep live R. parkeri contained, use a centrifuge with a sealed rotor that is designed to be removed from the centrifuge. This enables the transport of the sealed rotor in and out of the hood. 
			NOTE: As the R. parkeri are still alive before centrifugation, the sample needs to be contained until the rickettsia are killed. Thus, the centrifuge used to spin the R. parkeri culture onto the slide must have a sealed rotor that is designed to be lifted out of the centrifuge. With the rotor in the laminar flow hood, load the samples into the funnel-microscope slide assembly, reseal the rotor, and place the sealed rotor back into the centrifuge. Then, turn on the centrifuge, and the samples are deposited onto the microscope slides. After the run is finished, remove the sealed rotor from the centrifuge and place it in the laminar flow hood. There, open the rotor lid, and unload the funnel-microscope slide assembly inside the hood. Once dry, immerse the glass microscope slides in absolute methanol, which kills all pathogens. The funnels and the metal carriers are submerged in diluted DMQ, which kills R. parkeri.</li>
			<li>Air-dry the slides and fix in absolute methanol for 5 min at room temperature (RT).</li>
			<li>Stain the slides with Giemsa stain (4% in S&#248;renson&#39;s buffer, pH 6.6) for 30 min at 37 &#176;C and rinse with water for 5 s.</li>
			<li>Determine the infection percentage (number of cells infected with rickettsiae/100 cells) by light microscopy. 
			NOTE: Figure 1 shows typical Giemsa stain results.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Preparing cell-free R. parkeri 
	NOTE: When 90%-100% of the cells in the culture are infected, the rickettsiae should be isolated from the ISE6 cells and electroporation should be performed.
	<ol>
		<li>Add sterile 60-90 silicon carbide grit to sterile 2 mL microcentrifuge tubes to a volume of approximately 0.2 mL.</li>
		<li>Remove 2 mL of medium from 100% R. parkeri-infected cultures (step 1.1.3) and resuspend the cells in the remaining 3 mL of medium.</li>
		<li>Transfer equal portions of this suspension to tubes prepared in step 1.2.1.</li>
		<li>Vortex each tube at high speed for 30 s and then place the tubes on ice. Let the grit settle within seconds by gravity.</li>
		<li>In a class II biosafety cabinet, have a sterile 5 mL Luer-lock syringe ready with the plunger extended and the end of the plunger secured in a polystyrene base for 15 mL conical tubes.</li>
		<li>Using a sterile, 1 mL barrier pipet tip fitted to a suitable pipettor, remove the supernatant of the vortexed cell lysate from the tube, taking care not to aspirate any grit. Insert the pipet tip into the syringe hub opening and eject the contents into the syringe with gentle pressure. Alternatively, use a sterile, barrier 2 mL Pasteur pipette operated with a rubber bulb.</li>
		<li>Attach a sterilized 2 &#181;m pore size filter onto the syringe hub and filter the R. parkeri culture from step 1.2.6 into sterile 1.5 mL tubes.</li>
		<li>Collect the R. parkeri by centrifugation at 13,600 &#215; g at 4 &#176;C for 5 min, and discard the supernatant.</li>
		<li>Resuspend the pellet in 1.2 mL of ice-cold 300 mM sucrose and centrifuge again at 13,600 &#215; g at 4 &#176;C for 5 min. Repeat the resuspension and centrifugation for a total of two sucrose washes.</li>
		<li>Combine the R. parkeri pellets into one chilled, sterile 1.5 mL microcentrifuge tube in 50 &#181;L of ice-cold 300 mM sucrose per transformation. As one 25 cm2 flask of R. parkeri provides enough rickettsiae for two to three transformations, add 100-150 &#181;L of 300 mM cold sucrose. 
		NOTE: If desired, the number of rickettsiae can be calculated using a Petroff-Hausser counting chamber. When using an initial inoculation of 5 &#215; 107 -10 &#215; 107 cell-free R. parkeri, it will take 5-7 days on average to reach 90%-100% infection, yielding approximately 5 &#215; 107-10 &#215; 1010 cell-free R. parkeri.</li>
		<li>Gently resuspend the pellet by pipetting until it is thoroughly dispersed. Divide the sample into 50 &#181;L portions in chilled, sterile 1.5 mL microcentrifuge tubes. 
		NOTE: If desired, rickettsial viability may be assessed by flow cytometry following staining with a fluorescent dye 21.</li>
	</ol>
	</li>
</ol>
2. Transformation of R. parkeri with the pRAM18dSFA plasmid
<ol>
	<li>On ice, add 3 &#181;g of endotoxin-free pRAM18dSFA plasmid DNA13,15,20 to each tube containing 50 &#181;L of R. parkeri suspension and stir the mixture with the pipet tip gently but thoroughly. 
	NOTE: When preparing plasmid DNA, use a purification kit that includes an endotoxin-removal step to yield endotoxin-free plasmid DNA23. We found that a range of plasmid concentrations of between 1 &#181;g and 3 &#181;g per 50 &#181;L of R. parkeri suspension resulted in successful transformations, whereas increasing the amount of plasmid to 10 &#181;g inhibited transformation.</li>
	<li>Transfer the above mixture of DNA and R. parkeri into a chilled, sterile 0.1 cm gap electroporation cuvette (gently tap the cuvette until the mixture is evenly distributed) and let it sit on ice for 10-30 min.</li>
	<li>Remove the medium from a 100% confluent culture of ISE6 cells and resuspend the cell layers in 1.5 mL of fresh medium with NaHCO3 and HEPES buffer (described above in step 1.1.3). Use one flask of confluent cells per transformation.</li>
	<li>Transfer the resuspended cells to a sterile 2 mL microcentrifuge tube (one tube per 25 cm2 flask of ISE6 cells).</li>
	<li>Electroporate the R. parkeri/pRAM18dSFA mixture at 1.8 KV, 200 ohms, 25 &#181;F using an electroporator.</li>
	<li>Using a sterile, extended fine-tip transfer pipet, transfer a small amount of the ISE6 cell suspension (step 2.4) into the cuvette and gently pull the liquid up and down to wash out the cuvette. Transfer the mixture into the 2 mL microcentrifuge tube containing the remainder of the ISE6 cell suspension and gently mix the transformation mixture with the cells.</li>
	<li>Centrifuge the transformed samples at 700 &#215; g for 2 min at RT, and then increase the centrifugation speed to 13,600 &#215; g for 1 min more at RT. 
	NOTE: The two centrifugation speeds promote rickettsial binding with and entry into the cells: 700 &#215; g is used to pull down the ISE6 cells, while 13,600 &#215; g is used to pull down the rickettsiae.</li>
	<li>Let samples sit at RT or 34 &#176;C for 15 min to 1 h.</li>
	<li>Resuspend the transformants (two or three) in ISE6 cells with a sterile 2 mL barrier pipet tip fitted to a pipettor and transfer into two or three 25 cm2 cell culture flasks containing 3.5 mL of fresh medium with NaHCO3 and HEPES buffer. Alternatively, use a sterile, barrier 2 mL Pasteur pipette operated with a rubber bulb.</li>
	<li>Rock the flasks to spread the mixture evenly and then incubate at 34 &#176;C.</li>
	<li>After 16-24 h, add 10 &#181;L of spectinomycin (50 mg/mL) and 10 &#181;L of streptomycin (50 mg/mL) to each flask in step 2.10.</li>
</ol>
3. Observation of the transformed R. parkeri
NOTE: Use an epifluorescence microscope with rhodamine/TRITC filters to observe the flasks prepared in step 2.11 after 3-7 days. Once plaques are evident in the cultures (5-14 days), transformed R. parkeri can be seen that express the red fluorescent protein mKATE, encoded on the pRAM18dSFA plasmid.
<ol>
	<li>Confocal microscopy
	<ol>
		<li>Resuspend the cell cultures from the step 2.11 flasks and mix 100 &#181;L of these cell cultures with 5 &#181;L of Hoechst 33342 solution for 10-30 min at RT in the dark. 
		NOTE: Hoechst 33342 is a cell-permeable nuclear counterstain that binds to live tick cell DNA and emits blue fluorescence.</li>
		<li>Centrifuge 50 &#181;L of the mixture onto glass microscope slides using a centrifuge at 5 &#215; g for 3 min.</li>
		<li>Add 3 &#181;L of 1x PBS to the spot of cells deposited on the slide, overlay with a cover slip, and view with a confocal microscope (60x objective). Use the following excitation and emission parameters for fluorescent imaging: for 4&#8242;,6-diamidino-2-phenylindole (DAPI), excitation at 350 nm, emission at 470 nm; for tetramethylrhodamine (TRITC), excitation at 557 nm, emission at 576 nm.</li>
	</ol>
	</li>
</ol>

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No conflicts of interest are declared.

Here, we demonstrate a method for introducing exogenous DNA encoded on the shuttle plasmid pRAM18dSFA into rickettsiae using electroporation. In this procedure, cell-free rickettsiae were purified from host cells, transformed with a rickettsial shuttle vector, and released onto tick cells for infection. Also described is a confocal immunofluorescence procedure to detect red fluorescence protein-expressing R. parkeri in tick cells. Similar methods are applicable to other Rickettsia species and with furth...

Rickettsioses are caused by a broad range of obligate intracellular bacteria that belong to the genus Rickettsia (family Rickettsiaceae, order Rickettsiales). The genus Rickettsia is classified into four major groups based on phylogenetic characteristics1,2: the spotted fever group (SFG), which contains those rickettsiae that cause the most severe and fatal tick-borne rickettsioses (e.g., Rickettsia rickettsii, the causative agent of Rocky Mountain Spotted Fever), the typhus group (TG, e.g., Rickettsia prowazekii, the agent of epidemic typhus), the transitional group (TRG, e.g., Rickettsia felis, the causative agent of flea-borne spotted fever), and the ancestral group (AG, e.g., Rickettsia bellii).
Among the oldest known vector-borne diseases, rickettsioses are mainly acquired following transmission of the pathogens through the bites of infected arthropod vectors, including ticks, fleas, lice, and mites3,4. Although the discovery of effective antibiotics improved treatment outcomes, emerging and re-emerging epidemic rickettsioses continue to challenge traditional prevention and control strategies. Thus, a comprehensive understanding of rickettsia/host/vector interactions would ultimately establish a strong foundation for developing new approaches to prevent and cure these ancient diseases.
In nature, horizontal gene transfer (HGT) in bacteria occurs through conjugation, transduction, and transformation5. In vitro bacterial transformation utilizes these HGT concepts, although the intracellular nature of rickettsiae presents some challenges. The restricted growth conditions and poorly understood conjugation and transduction systems in different species of rickettsiae have prevented the application of conjugation and transduction methods in rickettsiae6,7,8. Compared with other obligate intracellular bacterial genera (e.g., Chlamydia, Coxiella, Anaplasma, and Ehrlichia), the genus Rickettsia differs with regard to the growth and replication strategies within the cell cytoplasm, which imposes specific challenges to the genetic modification of rickettsiae due to their unique lifestyle features9.
The initial hurdle to overcome when attempting the genetic modification of rickettsiae is to achieve successful transformation. Thus, designing a feasible approach with high transformation efficiency would be extremely valuable for developing genetic tools for rickettsiae. Here, we focus on electroporation, a broadly recognized transformation method that has been used to introduce exogenous DNA successfully into several species of rickettsiae, including Rickettsia prowazekii, Rickettsia typhi, Rickettsia conorii, Rickettsia parkeri, Rickettsia montanensis, Rickettsia bellii, Rickettsia peacockii, and Rickettsia buchneri10,11,12,13,14,15,16.
This paper describes a procedure for the electroporation of the R. parkeri strain Tate&#39;s Hell (accession: GCA_000965145.1) with the shuttle vector pRAM18dSFA derived from the Rickettsia amblyommatis strain AaR/SC plasmid pRAM18 engineered to encode mKATE, a far-red fluorescent protein, and aadA, conferring spectinomycin and streptomycin resistance13,15,20. Transformed R. parkeri are viable and stably maintained under antibiotic selection in tick cell lines. In addition, we show that the localization of transformed R. parkeri in live tick cells via confocal microscopy can be used to assess the quality of transformation rates in vector cell lines.

<table><tbody><tr><td>0.1 cm gap gene pulser electroporation cuvette</td><td>Bio-Rad</td><td>1652083</td><td></td></tr><tr><td>2 &amp;mu;m pore size filter&amp;nbsp;</td><td>GE Healthcare Life Sciences Whatman</td><td>6783-2520</td><td></td></tr><tr><td>5 mL Luer-lock syringe&amp;nbsp;</td><td>BD</td><td>309646</td><td></td></tr><tr><td>60-90 silicon carbide grit&amp;nbsp;</td><td>LORTONE, inc</td><td>&amp;nbsp;591-056</td><td></td></tr><tr><td>absolute methanol&amp;nbsp;</td><td>Fisher Scientific</td><td>A457-4</td><td></td></tr><tr><td>Bacto tryptose phosphate broth&amp;nbsp;</td><td>BD</td><td>260300</td><td></td></tr><tr><td>Cytospin centrifuge Cytospin4</td><td>Thermo Fisher Scientific</td><td>A78300003</td><td>The rotor is detachable so the whole rotor can be put into the hood to load infectious samples</td></tr><tr><td>EndoFree Plasmid Maxi Kit (10)</td><td>QIAGEN</td><td>12362</td><td>used to obtain endotoxin-free pRAM18dSFA plasmid</td></tr><tr><td>extended fine tip transfer pipet&amp;nbsp;</td><td>Perfector Scientific</td><td>TP03-5301</td><td></td></tr><tr><td>fetal bovine serum&amp;nbsp;</td><td>Gemini Bio</td><td>900-108</td><td>The FBS batch has to be tested to make sure ISE6 cells will grow well in it.</td></tr><tr><td>Gene Pulser II electroporator with Pulse Controller PLUS</td><td>Bio-Rad</td><td>165-2105 &amp;amp; 165-2110</td><td></td></tr><tr><td>hemocytometer</td><td>Thermo Fisher Scientific</td><td>267110</td><td></td></tr><tr><td>HEPES</td><td>Millipore-Sigma</td><td>H4034</td><td></td></tr><tr><td>ImageJ Fiji</td><td>National Institute of Health</td><td></td><td>raw image editing</td></tr><tr><td>KaryoMAX Giemsa stain&amp;nbsp;</td><td>Gibco</td><td>&amp;nbsp;2021-10-30</td><td></td></tr><tr><td>Leibovitz&#039;s L-15 medium</td><td>Gibco</td><td>41300039</td><td></td></tr><tr><td>lipoprotein concentrate&amp;nbsp;</td><td>MP Biomedicals</td><td>191476</td><td></td></tr><tr><td>Nikon Diaphot</td><td>Nikon</td><td></td><td>epifluorescence microscope</td></tr><tr><td>NucBlue Live ReadyProbes Reagent&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>R37605</td><td></td></tr><tr><td>Olympus Disc Scanning Unit (DSU) confocal microscope&amp;nbsp;</td><td>Olympus</td><td></td><td></td></tr><tr><td>Petroff-Hausser Counting Chamber</td><td>Hausser Scientific</td><td>&amp;nbsp;Chamber 3900</td><td></td></tr><tr><td>sodium bicarbonate</td><td>Millipore-Sigma</td><td>S5761</td><td></td></tr><tr><td>Vortex</td><td>Fisher Vortex Genie 2</td><td>12-812</td><td></td></tr></tbody></table>

an electroporation method to transform rickettsia spp. with a fluorescent protein-expressing shuttle vector in tick cell lines

Rickettsioses are caused by a broad range of obligate intracellular bacteria belonging to the genus Rickettsia that can be transmitted to vertebrate hosts through the bite of infected arthropod vectors. To date, emerging or re-emerging epidemic rickettsioses remain a public health risk due to the difficulty in diagnosis, as diagnostic methods are limited and not standardized or universally accessible. Misdiagnosis resulting from a lack of recognition of the signs and symptoms may result in delayed antibiotic treatment and poor health outcomes. A comprehensive understanding of Rickettsia characteristics would ultimately improve clinical diagnosis, assessment, and treatment with improved control and prevention of the disease. 
Functional studies of rickettsial genes are crucial for understanding their role in pathogenesis. This paper describes a procedure for the electroporation of the Rickettsia parkeri strain Tate's Hell with the shuttle vector pRAM18dSFA and the selection of transformed R. parkeri in tick cell culture with antibiotics (spectinomycin and streptomycin). A method is also described for the localization of transformed R. parkeri in tick cells using confocal immunofluorescence microscopy, a useful technique for checking transformation in vector cell lines. Similar approaches are also suitable for the transformation of other rickettsiae.

The morphology of R. parkeri in ISE6 cells under a light microscope after Giemsa staining are shown in Figure 1. In Figure 2, transformed R. parkeri expressing red fluorescence protein in ISE6 cells are shown using confocal microscopy. There is a substantial increase in the infection rate of transformed R. parkeri (red) in ISE6 cells (blue, corresponds to the nuclei) from (A) day 7 to (B) day 10 of inc...

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An Electroporation Method to Transform Rickettsia spp. with a Fluorescent Protein-Expressing Shuttle Vector in Tick Cell Lines

Electroporation is a rapid, broadly adopted method for introducing exogenous DNA into the genus Rickettsia. This protocol provides a useful electroporation method for the transformation of obligate intracellular bacteria in the genus Rickettsia.

An Electroporation Method to Transform Rickettsia spp. with a Fluorescent Protein-Expressing Shuttle Vector in Tick Cell Lines

Rickettsioses are caused by a broad range of obligate intracellular bacteria belonging to the genus Rickettsia that can be transmitted to vertebrate hosts ...

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1.8K Views.  University of Minnesota. Functionele studies van Rickettsia-genen zijn cruciaal voor het begrijpen van hun rol in pathogenese, dus we hebben betrouwbare methoden nodig om de genen op een gecontroleerde manier te veranderen. Ons protocol maakt gebruik van elektroporatie om exogene DNA te introduceren in obligate intracellulaire bacteriën van het geslacht Rickettsia, waardoor we de effecten van het geïntroduceerde DNA kunnen bestuderen. Lisa Price, een onderzoeker van Dr.Munderloh en het laboratorium van Dr.Kurtti, d...

Video: An Electroporation Method to Transform Rickettsia spp. with a Fluorescent Protein-Expressing Shuttle Vector in Tick Cell Lines

RNA Interference in Ticks

Ticks are obligate hematophagous ectoparasites of wild and domestic animals and humans, and are considered to be second worldwide to mosquitoes as vectors of human diseases1 and the most important vectors affecting cattle industry worldwide2. Ticks are classified in the subclass Acari, order Parasitiformes, suborder Ixodida and are distributed worldwide from Arctic to tropical regions3. Despite efforts to control tick infestations, these ectoparasites remain a serious problem for human and animal health4,5.
RNA interference (RNAi)6 is a nucleic acid-based reverse genetic approach that involves disruption of gene expression in order to determine gene function or its effect on a metabolic pathway. Small interfering RNAs (siRNAs) are the effector molecules of the RNAi pathway that is initiated by double-stranded RNA (dsRNA) and results in a potent sequence-specific degradation of cytoplasmic mRNAs containing the same sequence as the dsRNA trigger7-9. Post-transcriptional gene silencing mechanisms initiated by dsRNA have been discovered in all eukaryotes studied thus far, and RNAi has been rapidly developed in a variety of organisms as a tool for functional genomics studies and other applications10.
RNAi has become the most widely used gene-silencing technique in ticks and other organisms where alternative approaches for genetic manipulation are not available or are unreliable5,11. The genetic characterization of ticks has been limited until the recent application of RNAi12,13. In the short time that RNAi has been available, it has proved to be a valuable tool for studying tick gene function, the characterization of the tick-pathogen interface and the screening and characterization of tick protective antigens14. Herein, a method for RNAi through injection of dsRNA into unfed ticks is described. It is likely that the knowledge gained from this experimental approach will contribute markedly to the understanding of basic biological systems and the development of vaccines to control tick infestations and prevent transmission of tick-borne pathogens15-19.

A method for RNA interference (RNAi) by injection of dsRNA into unfed ticks is described. RNAi is the most widely used gene-silencing technique in ticks where the use of other methods of genetic manipulation has been limited.

Ticks are obligate hematophagous ectoparasites of wild and domestic animals and humans, and are considered to be second worldwide to mosquitoes as vectors ...

Immunology and Infection

Methods for Rapid Transfer and Localization of Lyme Disease Pathogens Within the Tick Gut

Lyme disease is caused by infection with the spirochete pathogen Borrelia burgdorferi, which is maintained in nature by a tick-rodent infection cycle 1. A tick-borne murine model 2 has been developed to study Lyme disease in the laboratory. While na&iacute;ve ticks can be infected with B. burgdorferi by feeding them on infected mice, the molting process takes several weeks to months to complete. Therefore, development of more rapid and efficient tick infection techniques, such as a microinjection-based procedure, is an important tool for the study of Lyme disease 3,4. The procedure requires only hours to generate infected ticks and allows control over the delivery of equal quantities of spirochetes in a cohort of ticks. This is particularly important as the generation of B. burgdorferi infected ticks by the natural feeding process using mice fails to ensure 100% infection rate and potentially results in variation of pathogen burden amongst fed ticks. Furthermore, microinjection can be used to infect ticks with B. burgdorferi isolates in cases where an attenuated strain is unable to establish infection in mice and thus can not be naturally acquired by ticks 5. This technique can also be used to deliver a variety of other biological materials into ticks, for example, specific antibodies or double stranded RNA 6. In this article, we will demonstrate the microinjection of nymphal ticks with in vitro-grown B. burgdorferi. We will also describe a method for localization of Lyme disease pathogens in the tick gut using confocal immunofluorescence microscopy.

Lyme disease research studies often require generation of ticks infected with the pathogen Borrelia burgdorferi, a process that typically takes several weeks. Here we demonstrate a microinjection-based tick infection procedure that can be accomplished within hours. We also demonstrate an immunofluorescence method for in situ localization of B. burgdorferi within ticks.

Lyme disease is caused by infection with the spirochete pathogen Borrelia burgdorferi, which is maintained in nature by a tick-rodent infection cycle 1.  ...

Purification of Biotinylated Cell Surface Proteins from Rhipicephalus microplus Epithelial Gut Cells

Rhipicephalus microplus &#8211; the cattle tick &#8211; is the most significant ectoparasite in terms of economic impact on livestock as a vector of several pathogens. Efforts have been dedicated to the cattle tick control to diminish its deleterious effects, with focus on the discovery of vaccine candidates, such as BM86, located on the surface of the tick gut epithelial cells. Current research focuses upon the utilization of cDNA and genomic libraries, to screen for other vaccine candidates. The isolation of tick gut cells constitutes an important advantage in investigating the composition of surface proteins upon the tick gut cells membrane. This paper constitutes a novel and feasible method for the isolation of epithelial cells, from the tick gut contents of semi-engorged R. microplus. This protocol utilizes TCEP and EDTA to release the epithelial cells from the subepithelial support tissues and a discontinuous density centrifugation gradient to separate epithelial cells from other cell types. Cell surface proteins were biotinylated and isolated from the tick gut epithelial cells, using streptavidin-linked magnetic beads allowing for downstream applications in FACS or LC-MS/MS-analysis.

Purification of Biotinylated Cell Surface Proteins from Rhipicephalus microplus Epithelial Gut Cells

A modified density centrifugation gradient-based methodology was utilized to isolate epithelial cells from Rhipicephalus microplus gut tissue. Surface-bound proteins were biotinylated and purified through streptavidin magnetic beads for utilization in downstream applications.

Rhipicephalus microplus &#8211; the cattle tick &#8211; is the most significant ectoparasite in terms of economic impact on livestock as a vector of ...

Biochemistry

Electroporation of Functional Bacterial Effectors into Mammalian Cells

The study of protein interactions in the context of living cells can generate critical information about localization, dynamics, and interacting partners. This information is particularly valuable in the context of host-pathogen interactions. Many pathogen proteins function within host cells in a variety of way such as, enabling evasion of the host immune system and survival within the intracellular environment. To study these pathogen-protein host-cell interactions, several approaches are commonly used, including: in vivo infection with a strain expressing a tagged or mutant protein, or introduction of pathogen genes via transfection or transduction. Each of these approaches has advantages and disadvantages. We sought a means to directly introduce exogenous proteins into cells. Electroporation is commonly used to introduce nucleic acids into cells, but has been more rarely applied to proteins although the biophysical basis is exactly the same. A standard electroporator was used to introduce affinity-tagged bacterial effectors into mammalian cells. Human epithelial and mouse macrophage cells were cultured by traditional methods, detached, and placed in 0.4 cm gap electroporation cuvettes with an exogenous bacterial pathogen protein of interest (e.g. Salmonella Typhimurium GtgE). After electroporation (0.3 kV) and a short (4 hr) recovery period, intracellular protein was verified by fluorescently labeling the protein via its affinity tag and examining spatial and temporal distribution by confocal microscopy. The electroporated protein was also shown to be functional inside the cell and capable of correct subcellular trafficking and protein-protein interaction. While the exogenous proteins tended to accumulate on the surface of the cells, the electroporated samples had large increases in intracellular effector concentration relative to incubation alone. The protocol is simple and fast enough to be done in a parallel fashion, allowing for high-throughput characterization of pathogen proteins in host cells including subcellular targeting and function of virulence proteins.

Electroporation was used to insert purified bacterial virulence effector proteins directly into living eukaryotic cells. Protein localization was monitored by confocal immunofluorescence microscopy. This method allows for studies on trafficking, function, and protein-protein interactions using active exogenous proteins, avoiding the need for heterologous expression in eukaryotic cells.

The study of protein interactions in the context of living cells can generate critical information about localization, dynamics, and interacting partners. ...

Rapid Protocol for Preparation of Electrocompetent Escherichia coli and Vibrio cholerae

Electroporation has become a widely used method for rapidly and efficiently introducing foreign DNA into a wide range of cells. Electrotransformation has become the method of choice for introducing DNA into prokaryotes that are not naturally competent. Electroporation is a rapid, efficient, and streamlined transformation method that, in addition to purified DNA and competent bacteria, requires commercially available gene pulse controller and cuvettes. In contrast to the pulsing step, preparation of electrocompetent cells is time consuming and labor intensive involving repeated rounds of centrifugation and washes in decreasing volumes of sterile, cold water, or non-ionic buffers of large volumes of cultures grown to mid-logarithmic phase of growth. Time and effort can be saved by purchasing electrocompetent cells from commercial sources, but the selection is limited to commonly employed E. coli laboratory strains. We are hereby disseminating a rapid and efficient method for preparing electrocompetent E. coli, which has been in use by bacteriology laboratories for some time, can be adapted to V. cholerae and other prokaryotes. While we cannot ascertain whom to credit for developing the original technique, we are hereby making it available to the scientific community.

Rapid Protocol for Preparation of Electrocompetent Escherichia coli and Vibrio cholerae

Electroporation is a commonly employed method for introducing DNA into bacteria in a process known as transformation. Traditional protocols for the preparation of electrocompetent cells are time consuming and labor intensive. This article describes an alternate, rapid, and efficient method for the preparation of electrocompetent cells presently employed by some laboratories.

Electroporation has become a widely used method for rapidly and efficiently introducing foreign DNA into a wide range of cells. Electrotransformation has ...

Phage-Mediated Genetic Manipulation of the Lyme Disease Spirochete Borrelia burgdorferi

Introducing foreign DNA into the spirochete Borrelia burgdorferi has been almost exclusively accomplished by transformation using electroporation. This process has notably lower efficiencies in the Lyme disease spirochete relative to other, better-characterized Gram-negative bacteria. The rate of success of transformation is highly dependent upon having concentrated amounts of high-quality DNA from specific backgrounds and is subject to significant strain-to-strain variability. Alternative means for introducing foreign DNA (i.e., shuttle vectors, fluorescent reporters, and antibiotic-resistance markers) into B. burgdorferi could be an important addition to the armamentarium of useful tools for the genetic manipulation of the Lyme disease spirochete. Bacteriophage have been well-recognized as natural mechanisms for the movement of DNA among bacteria in a process called transduction. In this study, a method has been developed for using the ubiquitous borrelial phage &#966;BB-1 to transduce DNA between B. burgdorferi cells of both the same and different genetic backgrounds. The transduced DNA includes both borrelial DNA and heterologous DNA in the form of small shuttle vectors. This demonstration suggests a potential use of phage-mediated transduction as a complement to electroporation for the genetic manipulation of the Lyme disease spirochete. This report describes methods for the induction and purification of phage &#966;BB-1 from B. burgdorferi, the use of this phage in transduction assays, and the selection and screening of potential transductants.

Phage-Mediated Genetic Manipulation of the Lyme Disease Spirochete Borrelia burgdorferi

The ability of bacteriophage to move DNA between bacterial cells makes them effective tools for the genetic manipulation of their bacterial hosts. Presented here is a methodology for inducing, recovering, and using &#966;BB-1, a bacteriophage of Borrelia burgdorferi, to transduce heterologous DNA between different strains of the Lyme disease spirochete.

Introducing foreign DNA into the spirochete Borrelia burgdorferi has been almost exclusively accomplished by transformation using electroporation. This ...

Internalization and Observation of Fluorescent Biomolecules in Living Microorganisms via Electroporation

The ability to study biomolecules in vivo is crucial for understanding their function in a biological context. One powerful approach involves fusing molecules of interest to fluorescent proteins such as GFP to study their expression, localization and function. However, GFP and its derivatives are significantly larger and less photostable than organic fluorophores generally used for in vitro experiments, and this can limit the scope of investigation. 
We recently introduced a straightforward, versatile and high-throughput method based on electroporation, allowing the internalization of biomolecules labeled with organic fluorophores into living microorganisms. Here we describe how to use electroporation to internalize labeled DNA fragments or proteins into Escherichia coli and Saccharomyces cerevisi&#230;, how to quantify the number of internalized molecules using fluorescence microscopy, and how to quantify the viability of electroporated cells. Data can be acquired at the single-cell or single-molecule level using fluorescence or FRET. The possibility of internalizing non-labeled molecules that trigger a physiological observable response in vivo is also presented. Finally, strategies of optimization of the protocol for specific biological systems are discussed.

Studies of biomolecules in vivo are crucial for understanding molecular function in a biological context. Here we describe a novel method allowing the internalization of fluorescent biomolecules, such as DNA or proteins, into living microorganisms. Analysis of in vivo data recorded by fluorescence microscopy is also presented and discussed.

The ability to study biomolecules in vivo is crucial for understanding their function in a biological context. One powerful approach involves fusing ...

Embryo Injection Technique for Gene Editing in the Black-Legged Tick, Ixodes scapularis

Ticks can transmit various viral, bacterial, and protozoan pathogens and are therefore considered vectors of medical and veterinary importance. Despite the growing burden of tick-borne diseases, research on ticks has lagged behind insect disease vectors due to challenges in applying genetic transformation tools for functional studies to the unique biology of ticks. Genetic interventions have been gaining attention to reduce mosquito-borne diseases. However, the development of such interventions requires stable germline transformation by injecting embryos. Such an embryo injection technique is lacking for chelicerates, including ticks. Several factors, such as an external thick wax layer on tick embryos, hard chorion, and high intra-oval pressure, are some&#160;obstacles that previously prevented embryo injection protocol development in ticks. The present work has overcome these obstacles, and an embryo injection technique for the black-legged tick, Ixodes scapularis, is described here.&#160;This technique can be used to deliver components, such as CRISPR/Cas9, for stable germline transformations.

Embryo Injection Technique for Gene Editing in the Black-Legged Tick, Ixodes scapularis

The present protocol describes a method for injecting tick embryos. Embryo injection is the preferred technique for genetic manipulation to generate transgenic lines.

Ticks can transmit various viral, bacterial, and protozoan pathogens and are therefore considered vectors of medical and veterinary importance. Despite ...

Bacterial Transformation: Electroporation

The term &#x201C;transformation&#x201D; refers cellular ingestion of foreign DNA. In nature, transformation can occur in certain types of bacteria. In molecular biology, however, transformation is artificially induced through the creation of pores in the bacterial cell walls. Bacterial cells that are able to take up DNA from the environment are called competent cells. Electrocompetent cells can be produced in the laboratory and transformation of these cells can be achieve via the application of an electrical field that creates pores in the cell wall through which DNA can pass.
The video explains the equipment used in electroporation such as an electroporator and electroporation cuvette. The video also goes through a step-by-step procedure about how to create electrocompetent cells and electroporate cells of interest. 
Prediction of the success of a transformation of an experiment, by observing the time constant, as well as the importance of removing salt from the solutions when electroporating, are also mentioned.

The term &#x201C;transformation&#x201D; refers cellular ingestion of foreign DNA.  In nature, transformation can occur in certain types of bacteria.  In ...

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Electroporation-Mediated In Vitro Gene Delivery: An Electric Pulse Technique to Introduce Fluorescent Reporter Protein-Encoding Plasmids Into Cultured Cells

Source: Lo, J. C. W. et al., <a href="https://www.jove.com/v/63283">Efficient and Cost Effective Electroporation Method to Study Primary Cilium-Dependent Signaling Pathways in the Granule Cell Precursor.</a>&#160;J. Vis. Exp.&#160; (2021)
This video demonstrates the electroporation technique that introduces fluorescent protein-encoding plasmid DNA into mouse primary cerebellar granule cell precursors.

Source: Lo, J. C. W. et al., Efficient and Cost Effective Electroporation Method to Study Primary Cilium-Dependent Signaling Pathways in the Granule Cell ...

An Electroporation Method to Transform Rickettsia spp. with a Fluorescent Protein-Expressing Shuttle Vector in Tick Cell Lines

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