This work was supported by the National Key Research and Development Program of China (2017YFD0501200) and the National Natural Science Foundation of China (31572507, 31772728 and 31873007).

Chickens for all animal experiments were housed and maintained according to the China Agricultural University Institutional Animal Care and Use Committee guidelines and followed the International Guiding Principles for Biomedical Research Involving Animals. The experiments were approved by the Beijing Administration Committee of Laboratory Animals.
1. Extraction and purification of sporozoites of Eimeria spp. (e.g., E. tenella)
<ol>
	<li>Release of sporocysts
	<ol>
		<li>Centrifuge 1 x 107sporulated oocysts in potassium dichromate solution (2.5%, m/v) at 2,300 x g for 5 min. Wash them with PBS (phosphate buffer solution) three times.</li>
		<li>Resuspend the pellets with 1 mL of PBS and transfer to a 15 mL tube. Add an equal volume of glass beads (1 mm x 1 mm diameter range) and oscillate the oocyst suspension using a vortex mixer to release the sporocysts.</li>
		<li>Monitor the release of sporocysts by microscopy every minute. Stop vortexing when more than 90% of oocysts are broken. 
		NOTE: Most of the oocysts (such as E. tenella, E. necatrix, and E. acervulina) were broken after 1 min using the vortex mixer.</li>
		<li>Transfer the sporocyst suspension to new 1.5 mL tubes and centrifuge at 1,600 x g for 5 min.</li>
		<li>Resuspend the precipitate with 1 mL of 50% density gradient solution, combine in a 1.5 mL tube, and centrifuge at 10,000 x g for 1 min. 
		NOTE: For the density gradient composition, refer to Table 1. The density gradient is a silica-based colloidal medium, consisting of colloidal silica particles of 15-30 nm diameter (23% w/w in water), which have been coated using polyvinylpyrrolidone (PVP).</li>
	</ol>
	</li>
	<li>Release of sporozoites
	<ol>
		<li>Resuspend the precipitate with the excystation buffer (Table 1) and incubate in a 42 &#176;C water bath for 40-60 min to release the sporozoites. Stop incubating when more than 90% of sporozoites are released. Then centrifuge at 600 x g for 10 min. 
		NOTE: Shake the tubes once every 5 minutes during excystation.</li>
		<li>Resuspend the precipitation with 1 mL of 55% density gradient solution and centrifuge at 10,000 x g for 1 min.</li>
		<li>Resuspend the precipitation with 1 mL of PBS and count the sporozoites using a hemocytometer.</li>
	</ol>
	</li>
</ol>
2. Collection and purification of merozoites of E. necatrix
NOTE: Use Arbor Acre (AA) broilers aged 7-14 d in the experiment. Coccidia-free chickens (n=3) were inoculated with 2 x 105 oocysts of E. necatrix. At 109 h post-infection, the birds were sacrificed by cervical dislocation. The intestine was removed for the collection of the 2nd generation merozoites. For different Eimeria species, there was a different time for collection of the 2nd generation merozoites: E. necatrix at 109 h, and E. tenella at 112 h post-inoculation. For the transfection of E. necatrix, merozoites are the optimal choice as the second merozoites are easy to purify.
<ol>
	<li>Collection of the second-generation merozoites of E. necatrix
	<ol>
		<li>Cut the chicken intestine longitudinally, from the yolk stalk(the middle of the small intestine) to the ileocecal orifice, and wash it with PBS or HBSS (Hank&#39;s Balanced Salt Solution) gently three times in a Petri dish.</li>
		<li>Cut the intestine into 0.5 cm x 0.5 cm pieces and place it in a conical flask with a digestion buffer (Table 1). Place the flask on a magnetic mixer at 37 &#176;C with a stirring bar for 30-60 min to release merozoites. After 30 min of incubation, monitor the release of merozoites by microscopic examination every 5 min.</li>
	</ol>
	</li>
	<li>Purify the merozoites by filtration and centrifugation.
	<ol>
		<li>Filter the suspension containing digested merozoites using four layers of gauze14, and centrifuge at 600 x g for 10 min.</li>
		<li>After centrifugation, discard the supernatant containing intestine debris. Transfer the precipitation with purified merozoites to 1.5 mL tubes.</li>
		<li>Resuspend the precipitation with 1 mL of PBS and count the merozoites using a hemocytometer.</li>
	</ol>
	</li>
</ol>
3. Nucleofection of merozoites or sporozoites
<ol>
	<li>Preparation before nucleofection of parasites
	<ol>
		<li>Prepare about 107 merozoites or sporozoites in one tube. If transfecting merozoites, prepare 3-4 tubes.</li>
		<li>Prepare an amount of plasmid DNA or purified PCR fragment that is greater than or equal to 10 &#181;g. 
		NOTE: The plasmid used in this study contains 2 genes: enhanced yellow fluorescent protein (EYFP) and dihydrofolate reductase thymidylate synthase derived from Toxoplasma gondii (TgDHFR-TS)15.</li>
		<li>Prepare 25 U of restriction enzyme. If plasmids are linearized, the restriction enzyme can improve the transfection efficiency. If the plasmids are circular, omit the restriction enzyme.</li>
		<li>Prepare 85 &#181;L of nucleofection buffer: mix 20 &#181;L of nucleofection buffer I and 1 mL of nucleofection buffer II, and use a part of the solution. The volume of the total buffer is 100 &#181;L.</li>
	</ol>
	</li>
	<li>Nucleofection
	<ol>
		<li>Centrifuge the sporozoite or merozoite suspension at 600 x g for 10 min. Then discard the supernatant.</li>
		<li>In the following order, add 85 &#181;L of nuclear transfection buffer, 10 &#181;g of plasmid (PCR fragment), and 25 U of restriction enzyme (usually 5 &#181;L) into the 1.5 mL tube containing sporozoites or merozoites.</li>
		<li>Transfer the suspension to a nuclear transfection cup. Put the cup into a nuclear transfer groove.</li>
		<li>Turn on the nucleofection device by using the power button and select the transfection procedure U-033. If the nucleofection device starts in the Free Program Choice mode, exit this mode by pressing the X button.</li>
		<li>When the program finishes, press the X button of the nucleofection device, and the screen should display OK, indicating that the nucleofection is successful.</li>
		<li>Add 0.5-1 mL of Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM)8 to the nucleofection cup to stop the reaction and transfer the suspension to 1.5 mL tube after mixing gently.</li>
	</ol>
	</li>
</ol>
4. Cloacal inoculation or intravenous injection
<ol>
	<li>Inoculate the nucleofected parasites into 7-day-old chickens. Inoculate the merozoites of E. necatrix or the sporozoites of E. tenella via the cloacal route, but inoculate E. acervulina sporozoites via intravenous injection. Inoculate about 2 x 107 million sporozoites into each chicken, and incoluate merozoites 107 for each bird.</li>
</ol>
5. Propagation and FACS sorting
<ol>
	<li>Collect oocysts from feces 5-9 days post-inoculation with transfected sporozoites. Collect the oocysts on the third day after inoculating with transfected merozoites.</li>
	<li>Use fluorescence-activated cell sorting (FACS) and 150 mg/kg pyrimethamine8 to successively increase the transgenic population ratio. 
	NOTE: Use pyrimethamine by adding it directly in the feed. For more convenient use, prepare water-soluble pyrimethamine. Dissolve 1 g of pyrimethamine in 0.2 mL pf H2SO4 and 9.8 mL of N-methyl pyrrolidone (NMP), and then add 1.5 mL of this stock solution into 1 L of drinking water for birds.</li>
</ol>
6. Optional column purification
NOTE: If more pure sporozoites or merozoites are needed, there is an optional method that purifies them through a diethylaminoethyl-52 cellulose (DE-52 cellulose) column.
<ol>
	<li>Prepare the DE-52 cellulose column at least one day in advance.
	<ol>
		<li>Prepare glycine eluent buffer (Table 1). Adjust the pH of glycine eluent buffer from 7.6 to 8.0 and prewarmed to 41 &#176;C.</li>
		<li>Add 2.5 g of DE-52 cellulose to the column. Add water and soak overnight. Discard the supernatant.</li>
		<li>Add water and soak for 1 h. Discard the supernatant.</li>
		<li>Add 0.1 M NaOH and soak for at least 2 hours. Repeat this step.</li>
		<li>Replace the supernatant with water. After the cellulose completely settles to the bottom (about half an hour), repeat this step.</li>
		<li>Discard the supernatant, add 0.1 M HCl, and soak for at least 2 hours. Repeat this step.</li>
		<li>Discard the supernatant, and soak the cellulose twice with glycine eluent buffer.</li>
		<li>Measure and adjust pH from 7.6 to 8.0 by adding 0.1 M HCl or 0.1 M NaOH. 
		NOTE: In this part, the liquid has at least 5x more volume than that of DE-52 cellulose.</li>
	</ol>
	</li>
	<li>Adjust the flow rate between 40-50 r/min.</li>
	<li>When the sedimentation of cellulose is completed, add the sporozoite or merozoite suspension to the chromatographic column. Adjust the flow rate to 30-40 r/min. 
	NOTE: Resuspend the sporozoite or merozoite precipitation with glycine eluent buffer before adding the chromatographic column.</li>
	<li>Collect with glycine eluent buffer into 50 mL tubes. Stop the collection according to the results of the microscopic examination of sporozoites or merozoites during the elution process.</li>
	<li>Centrifuge the glycine eluent buffer collected at 600 x g for 10 min. Transfer the sporozoite or merozoite precipitation to new 1.5 mL tubes.</li>
	<li>Count the sporozoites or merozoites using a hemocytometer.</li>
</ol>

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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+DNA+transformation+in+the+obligate+intracellular+parasite+Toxoplasma+gondii+by+complementation+of+tryptophan+auxotrophy.">Stable DNA transformation in the obligate intracellular parasite Toxoplasma gondii by complementation of tryptophan auxotrophy.</a> Proceedings of the National Academy of Sciences of the United States of America. 91 (12), 5508-5512 (1994).</li><li>Donald, R. G., Roos, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+molecular+transformation+of+Toxoplasma+gondii:+a+selectable+dihydrofolate+reductase-thymidylate+synthase+marker+based+on+drug-resistance+mutations+in+malaria.">Stable molecular transformation of Toxoplasma gondii: a selectable dihydrofolate reductase-thymidylate synthase marker based on drug-resistance mutations in malaria.</a> Proceedings of the National Academy of Sciences of the United States of America. 90 (24), 11703-11707 (1993).</li><li>Soldati, D., Boothroyd, J. 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C., 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=Stable+transfection+of+Eimeria+tenella:+Constitutive+expression+of+the+YFP-YFP+molecule+throughout+the+life+cycle.">Stable transfection of Eimeria tenella: Constitutive expression of the YFP-YFP molecule throughout the life cycle.</a> International Journal for Parasitology. 39 (1), 109-117 (2009).</li><li>Qin, M., 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=Transfection+of+Eimeria+mitis+with+Yellow+Fluorescent+Protein+as+Reporter+and+the+Endogenous+Development+of+the+Transgenic+Parasite.">Transfection of Eimeria mitis with Yellow Fluorescent Protein as Reporter and the Endogenous Development of the Transgenic Parasite.</a> PloS One. 9 (12), e114188 (2014).</li><li>Duan, C. 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D., 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=Eimeria+species+parasites+as+novel+vaccine+delivery+vectors:+anti-Campylobacter+jejuni+protective+immunity+induced+by+Eimeria+tenella-delivered+CjaA.">Eimeria species parasites as novel vaccine delivery vectors: anti-Campylobacter jejuni protective immunity induced by Eimeria tenella-delivered CjaA.</a> Vaccine. 30 (16), 2683-2688 (2012).</li><li>Eckert, J., Braun, R., Shirley, M. W., Coudert, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eimeria+species+and+strains+of+chickens.">Eimeria species and strains of chickens.</a> Biotechnology: Guidelines on techniques in coccidiosis research. Part. 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In the 1990s, a transfection system was developed for apicomplexan parasites, and it was used for studies on eimerian parasites. Recently, stable transfection was conducted in E. tenella8,9 and E. nieschulzi15. We achieved the stable transfection of E. necatrix by transfecting second-generation merozoites11. Inoculation of transfected sporozoites of E. acervulina through the wing ...

Eimeria spp. causes coccidiosis, which leads to substantial economic losses in the livestock and poultry industry. Although anticoccidial drugs, and to an extent, attenuated anticoccidial vaccines, have been used widely for the control of coccidiosis, there are still shortcomings regarding their drug resistance, drug residues, and the potential diffusion of vaccine strains that regain virulence1. With the development of molecular biology, transfection has become a vital tool for studying gene functions, developing novel vaccines, and screening new drug targets for Eimeria.
In the last decades, transfection has been applied successfully for apicomplexan parasites such as Plasmodium and Toxoplasma gondii2,3,4,5,6. A study using &#946;-gal as a reporter for the transfection in E. tenella piloted such work in Eimeria7. The transfection of E. tenella8,9, E. mitis10, and E. acervulina (Zhang et al., unpublished data) was successful in chickens. Recently, we achieved transfection using merozoites of E. necatrix through nucleofection11.
Studies showed that Eimeria expressing a heterologous antigen has the potential to be developed as a recombinant vaccine, such as those expressing Campylobacter jejuni antigen A (CjaA) or chicken interleukin 2 (chIL-2)12,13. Therefore, this protocol describes a nucleofection study of Eimeria spp. in chickens. The procedure describes purification of sporozoites or merozoites, nucleofection with plasmid DNA, cloacal inoculation/intravenous injection and in vivo propagation to help researchers starting studies on transgenic Eimeria parasites.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>ATP-disodium</td>
			<td>Sigma</td>
			<td>A26209</td>
			<td></td>
		</tr>
		<tr>
			<td>Cellulose DE-52</td>
			<td>Solarbio</td>
			<td>C8350</td>
			<td></td>
		</tr>
		<tr>
			<td>Constant Flow Pump</td>
			<td>SHANGHAI JINGKE INDUSTRIAL CO., LTD.</td>
			<td>HL-2B</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>MACGENE</td>
			<td>CM15019</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass beads</td>
			<td>Sigma</td>
			<td>Z250473-1PAK</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma</td>
			<td>No. V900116</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Biotopped</td>
			<td>G6200</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>MACGENE</td>
			<td>CC016</td>
			<td></td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Sigma</td>
			<td>No. V900041</td>
			<td></td>
		</tr>
		<tr>
			<td>Low Speed Centrifuge</td>
			<td>BEIJING ERA BEILI CENTRIFUGE CO., LTD.</td>
			<td>DT5-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic Mixer</td>
			<td>SCILOGEX</td>
			<td>MS-H280-Pro</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma</td>
			<td>449164</td>
			<td></td>
		</tr>
		<tr>
			<td>MoFlo cell sorter</td>
			<td>BeckMan Coulter, US</td>
			<td>201309995</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Sigma</td>
			<td>144-55-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Nucleofection device</td>
			<td>LONZA/amaxa</td>
			<td>90900012 (Nucleofector II)</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Solarbio</td>
			<td>P1010</td>
			<td></td>
		</tr>
		<tr>
			<td>Percoll (DG gradient stock solution)</td>
			<td>GE Healthcare</td>
			<td>17-0891-09</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium taurodeoxycholate hydrate</td>
			<td>Sigma</td>
			<td>T0875</td>
			<td></td>
		</tr>
		<tr>
			<td>Sorvall Legend Micro 17 Microcentrifuge</td>
			<td>ThermoFisher Scientific</td>
			<td>75002430</td>
			<td></td>
		</tr>
		<tr>
			<td>The composition of DMEM: 4.5 g/L glucose with sodium pyruvate, L-glutamine, and 25 mM HEPES.</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Solarbio</td>
			<td>T8150</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex Mixer</td>
			<td>Beijing North TZ-Biotech Develop.co.</td>
			<td>HQ-60-II</td>
			<td></td>
		</tr>
		<tr>
			<td>Water Bath Thermostat</td>
			<td>Grant Instruments (Cambridge), Ltd.</td>
			<td>GD120,GM0815010</td>
			<td></td>
		</tr>
	</tbody>
</table>

nucleofection and in vivo propagation of chicken eimeria parasites

Transfection is a technical process through which genetic material, such as DNA and double-stranded RNA, are delivered into cells to modify the gene of interest. Currently, transgenic technology is becoming an indispensable tool for the study of Eimeria, the causative agents of coccidiosis in poultry and livestock. This protocol provides a detailed description of stable transfection in eimerian parasites: purification and nucleofection of sporozoites or second-generation merozoites, and in vivo propagation of transfected parasites. Using this protocol, we achieved transfection in several species of Eimeria. Taken together, nucleofection is a useful tool to facilitate genetic manipulation in eimerian parasites.

This protocol has been used to transfect eimerian parasites. In this study, the 2nd generation meronts and merozoites of E. necatrix were shown in Figure 2A and Figure 2B, while Figure 2C and Figure 2D showed the sporocysts and sporozoites of E. tenella after using the densit...

Watch this Scientific Journal Video about Nucleofection and In Vivo Propagation of Chicken Eimeria Parasites at JoVE.com

Nucleofection and In Vivo Propagation of Chicken Eimeria Parasites

Here, we provided a method to achieve stable transfection of chicken Eimeria parasites by nucleofecting sporozoites or second-generation merozoites. Genetically modified eimerian parasites expressing heterologous antigenic genes could be used as vaccine delivery vehicles.

Nucleofection and In Vivo Propagation of Chicken Eimeria Parasites

Transfection is a technical process through which genetic material, such as DNA and double-stranded RNA, are delivered into cells to modify the gene of ...

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Research

JoVE Journal

Biology

6.8K Views.  China Agricultural University. Here, we provided a method to achieve stable transfection of chicken Eimeria parasites by nucleofecting sporozoites or second-generation merozoites. Genetically modified eimerian parasites expressing heterologous antigenic genes could be used as vaccine delivery vehicles.

Video: Nucleofection and In Vivo Propagation of Chicken Eimeria Parasites

Propagation of Human Embryonic Stem (ES) Cells

Propagation of Human Embryonic Stem ES Cells

Yeah, so I'm Lauren Deon. I'm in charge of the Human Amgen stem cell co facility at MGH. Yeah, so I'm Lauren, I'm in charge of the Human AM stem cell co facility at MGH.So I've been working with human Amgen stem cells for years now since I was a postdoc in George De Lab before, from four, four years. And so right now we have two cell lines, H one and H nine, which are both from Y cell. And so the way we are expanding or maintaining these cells in culture is by, at least the way we are splitting these cells is by using collagenous.So what I usually do is for the first few passage after throwing the cells, I use a technical, the picking colonies. So in this case, I can check under the microscope which colonies are good and differentiate and nice shape, and I will collect only these colonies and I will cut them in few pieces and put them back in, in a fresh flat. And the, the colonies that look kind of differentiate it are just left them alone.The other way to pass the cells is by using trypsin. So I don't use this technique here. I think collagenase is a little bit safer because human being stem cells don't like to be individualized or they don't like to be alone.So if you do trypsin, if you use trypsin for a little bit too long, if you use it for like three to four minutes, which is the way we use strips in for other type of cells, human stem cells will be individualized and most of them will die or just differentiate or will not attach to the myth. So the idea is you have to keep the cells as clumps. You don't want them to be alone in the in, in the flask.So using collagenase, the advantage is to keep cells as plants. Using chipsy, you just select for cells that will survive this very, very harsh process of g opsonization. So these cells are usually very highly proliferative.So you get, you can adapt your cells to this tripsin process. The problem is that you have cells that they behave differently, they go much faster. And one risk is that you can get some commercial abnormality to, to this, to this very high division process.So you say that ization can cause selection of cells in culture for most proliferative cells.Right. More ative and probably also higher survival rates than other cells. So after proliferation, basically culture after proliferation in tripsin, after several passages become a different culture, these different properties.So it's, it's hard to figure out if they are exactly identical. If you do the test, the usual test, let's say you check markers, regular markers of end differentiated cells like OC four, nano one 60 SC four C, C3, all this molecule might still be expressed. If you do TER formation, this cells might still differentiate and be formed like al like normal cells.If you look at the karyotype, you might have some abnormality, but you might still have a normal karyotype. The, the, the selective advantage might be due to just a mutation that you cannot see by doing a karyotype. So the fact that they grow much faster is by itself I think a little bit worser.And if we want to use these cells for transplantation at some point, I would definitely not use this type of cells.Okay. So would you like to tell me what are the main technical problems with using this technique for, for passages of cells? So using S, yes.It's just a little bit longer and also it's, since the cells are not going as fast, it's just much longer to get a high number of cells to do some experiment. So it's just less convenient I would say. Would you like to tell me about few words about the goals of your experiment?How do you use CS cells? Okay, so my main project here is to get endothelial cells from human AB stem cells. And the idea is to be able to make blood.And we are working a lot with tissue bioengineering labs and one of their goal is to make tissue in vitro. But one of the limitation to this is the lack of blood vessels going through this tissues. So if we can bring endothelial cells or put endothelial cells in this tissue, we would have better growth of this tissue.So the idea few sources of cells are known for endothelial cells like cold blood or pressure blood, but it would be interesting to have another source of cells like human years. And the advantage is since human years can be expanded and most infinitely, you could modify these cells, make some inducible system and be able to use it. Then in your final product, your material cells, in this case.

Windowing Chicken Eggs for Developmental Studies

The study of development has been greatly aided by the use of the chick embryo as an experimental model.  The ease of accessibility of the embryo has allowed for experiments to map cell fates using several approaches, including chick quail chimeras and focal dye labeling.  In addition, it allows for molecular perturbations of several types, including placement of protein-coated beads and introduction of plasmid DNA using in ovo electroporation.  These experiments have yielded important data on the development of the central and peripheral nervous systems.  For many of these studies, it is necessary to open the eggshell and reclose it without perturbing the embryo's growth.  The embryo can be examined at successive developmental stages by re-opening the eggshell.  While there are several excellent methods for opening chicken eggs, in this article we demonstrate one method that has been optimized for long survival times.  In this method, the egg rests on its side and a small window is cut in the shell.  After the experimental procedure, the shell is used to cover the egg for the duration of its development.  Clear plastic tape overlying the eggshell protects the embryo and helps retain hydration during the remainder of the incubation period.  This method has been used beginning at two days of incubation and has allowed survival through mature embryonic ages.

The ease of accessibility of the embryo has allowed for experiments to map cell fates using several approaches, including chick quail chimeras and focal dye labeling. In this article we demonstrate one egg preparation method that has been optimized for long survival times.

The study of development has been greatly aided by the use of the chick embryo as an experimental model.  The ease of accessibility of the embryo has ...

Placing Growth Factor-Coated Beads on Early Stage Chicken Embryos

The neural tube expresses many proteins in specific spatiotemporal patterns during development. These proteins have been shown to be critical for cell fate determination, cell migration, and formation of neural circuits. Neuronal induction and patterning involve bone morphogenetic protein (BMP), sonic hedgehog (SHH), fibroblast growth factor (FGF), among others. In particular, the expression pattern of Fgf8 is in close proximity to regions expressing BMP4 and SHH. This expression pattern is consistent with developmental interactions that facilitate patterning in the telencephalon.

Here we provide a visual demonstration of a method in which an in ovo preparation can be used to test the effects of Fgfs in the formation of the forebrain. Beads are coated with protein and placed in the developing neural tube to provide sustained exposure. Because the procedure uses small, carefully placed beads, it is minimally invasive and allows several beads to be placed within a single neural tube. Moreover, the method allows for continued development so that embryos can be analyzed at a more mature stage to detect changes in anatomy and in neural patterning. This simple but useful protocol allows for real time imaging. It provides a means to make spatially and temporally limited changes to endogenous protein levels.

A variety of growth factors and proteins interact to induce cells to take on different cell fates during development. Here we demonstrate the use of an in ovo preparation to address possible interactions between different proteins in development by placing beads on E2.5 chick embryos.

The neural tube expresses many proteins in specific spatiotemporal patterns during development.   These proteins have been shown to be critical for cell ...

In Ovo Electroporations of HH Stage 10 Chicken Embryos

Large size and external development of the chicken embryo have long made it a valuable tool in the study of developmental biology.  With the advent of molecular biological techniques, the chick has become a useful system in which to study gene regulation and function.  By electroporating DNA or RNA constructs into the developing chicken embryo, genes can be expressed or knocked down in order to analyze in vivo gene function.  Similarly, reporter constructs can be used for fate mapping or to examine putative gene regulatory elements.  Compared to similar experiments in mouse, chick electroporation has the advantages of being quick, easy and inexpensive.  This video demonstrates first how to make a window in the eggshell to manipulate the embryo.  Next, the embryo is visualized with a dilute solution of India ink injected below the embryo.  A glass needle and pipette are used to inject DNA and Fast Green dye into the developing neural tube, then platinum electrodes are placed parallel to the embryo and short electrical pulses are administered with a pulse generator.  Finally, the egg is sealed with tape and placed back into an incubator for further development.  Additionally, the video shows proper egg storage and handling and discusses possible causes of embryo loss following electroporation.

Chick in ovo electroporation is a technique which allows genetic manipulation of the avian embryo. Common applications of this technique include functional analysis of genes and putative enhancer elements. This video demonstrates neural tube electroporation in HH 10 chick embryos. Injection technique and proper egg handling are discussed.

Large size and external development of the chicken embryo have long made it a valuable tool in the study of developmental biology.  With the advent of ...

Xenotransplantation of Human Stem Cells into the Chicken Embryo

The chicken embryo is a classical animal model for studying normal embryonic and fetal development and for xenotransplantation experiments to study the behavior of cells in a standardized in vivo environment. The main advantages of the chicken embryo include low cost, high accessibility, ease of surgical manipulation and lack of a fully developed immune system. Xenotransplantation into chicken embryos can provide valuable information about cell proliferation, differentiation and behavior, the responses of cells to signals in defined embryonic tissue niches, and tumorigenic potential. Transplanting cells into chicken embryos can also be a step towards transplantation experiments in other animal models. Recently the chicken embryo has been used to evaluate the neurogenic potential of human stem and progenitor cells following implantation into neural anlage1-6. In this video we document the entire procedure for transplanting human stem cells into the developing central nervous system of the chicken embryo. The procedure starts with incubation of fertilized eggs until embryos of the desired age have developed. The eggshell is then opened, and the embryo contrasted by injecting dye between the embryo and the yolk. Small lesions are made in the neural tube using microsurgery, creating a regenerative site for cell deposition that promotes subsequent integration into the host tissue. We demonstrate injections of human stem cells into such lesions made in the part of the neural tube that forms the hindbrain and the spinal cord, and into the lumen of the part of the neural tube that forms the brain. Systemic injections into extraembryonic veins and arteries are also demonstrated as an alternative way to deliver cells to vascularized tissues including the central nervous system. Finally we show how to remove the embryo from the egg after several days of further development and how to dissect the spinal cord free for subsequent physiological, histological or biochemical analyses.

In this paper we present a method for transplanting human stem cells into various regions of the central nervous system of the chicken embryo. This provides an in vivo model for assessing the proliferation and differentiation of various types of human stem cells in embryonic tissue environments.

The chicken embryo is a classical animal model for studying normal embryonic and fetal development and for xenotransplantation experiments to study the ...

In vivo and in vitro Studies of Adaptor-clathrin Interaction

A major endocytic pathway initiates with the formation of clathrin-coated vesicles (CCVs) that transport cargo from the cell surface to endosomes1-6. CCVs are distinguished by a polyhedral lattice of clathrin that coats the vesicle membrane and serves as a mechanical scaffold. Clathrin coats are assembled during vesicle formation from individual clathrin triskelia , the soluble form of clathrin composed of three heavy and three light chain subunits7,8. Because the triskelion does not have the ability to bind to the membrane directly, clathrin-binding adaptors are critical to link the forming clathrin lattice to the membrane through association with lipids and/or membrane proteins9. Adaptors also package transmembrane protein cargo, such as receptors, and can interact with each other and with other components of the CCV formation machinery9.
Over twenty clathrin adaptors have been described, several are involved in clathrin mediated endocytosis and others localize to the trans Golgi network or endosomes9. With the exception of HIP1R (yeast Sla2p), all known clathrin adaptors bind to the N-terminal -propeller domain of the clathrin heavy chain9. Clathrin adaptors are modular proteins consisting of folded domains connected by unstructured flexible linkers. Within these linker regions, short binding motifs mediate interactions with the clathrin N-terminal domain or other components of the vesicle formation machinery9. Two distinct clathrin-binding motifs have been defined: the clathrin-box and the W-box9. The consensus clathrin-box sequence was originally defined as L[L/I][D/E/N][L/F][D/E]10 but variants have been subsequently discovered11. The W-box conforms to the sequence PWxxW (where x is any residue).
Sla1p (Synthetic Lethal with Actin binding protein-1) was originally identified as an actin associated protein and is necessary for normal actin cytoskeleton structure and dynamics at endocytic sites in yeast cells12. Sla1p also binds the NPFxD endocytic sorting signal and is critical for endocytosis of cargo bearing the NPFxD signal13,14. More recently, Sla1p was demonstrated to bind clathrin through a motif similar to the clathrin box, LLDLQ, termed a variant clathrin-box (vCB), and to function as an endocytic clathrin adaptor15. In addition, Sla1p has become a widely used marker for the endocytic coat in live cell fluorescence microscopy studies16. Here we use Sla1p as a model to describe approaches for adaptor-clathrin interaction studies. We focus on live cell fluorescence microscopy, GST-pull down, and co-immunoprecipitation methods.

In vivo and in vitro Studies of Adaptor-clathrin Interaction

Clathrin-mediated endocytosis depends on adaptor proteins that coordinate cargo selection and clathrin coat assembly. Here we describe procedures to study adaptor-clathrin physical interaction and live cell imaging approaches using as a model the yeast endocytic adaptor protein Sla1p.

A major endocytic pathway initiates with the formation of clathrin-coated vesicles (CCVs) that transport cargo from the cell surface to endosomes1-6. CCVs ...

Mechanical Stimulation-induced Calcium Wave Propagation in Cell Monolayers: The Example of Bovine Corneal Endothelial Cells

Intercellular communication is essential for the coordination of physiological processes between cells in a variety of organs and tissues, including the brain, liver, retina, cochlea and vasculature. In experimental settings, intercellular Ca2+-waves can be elicited by applying a mechanical stimulus to a single cell. This leads to the release of the intracellular signaling molecules IP3 and Ca2+ that initiate the propagation of the Ca2+-wave concentrically from the mechanically stimulated cell to the neighboring cells. The main molecular pathways that control intercellular Ca2+-wave propagation are provided by gap junction channels through the direct transfer of IP3 and by hemichannels through the release of ATP. Identification and characterization of the properties and regulation of different connexin and pannexin isoforms as gap junction channels and hemichannels are allowed by the quantification of the spread of the intercellular Ca2+-wave, siRNA, and the use of inhibitors of gap junction channels and hemichannels. Here, we describe a method to measure intercellular Ca2+-wave in monolayers of primary corneal endothelial cells loaded with Fluo4-AM in response to a controlled and localized mechanical stimulus provoked by an acute, short-lasting deformation of the cell as a result of touching the cell membrane with a micromanipulator-controlled glass micropipette with a tip diameter of less than 1 &mu;m. We also describe the isolation of primary bovine corneal endothelial cells and its use as model system to assess Cx43-hemichannel activity as the driven force for intercellular Ca2+-waves through the release of ATP. Finally, we discuss the use, advantages, limitations and alternatives of this method in the context of gap junction channel and hemichannel research.

Intercellular Ca2+-waves are driven by gap junction channels and hemichannels. Here, we describe a method to measure intercellular Ca2+-waves in cell monolayers in response to a local single-cell mechanical stimulus and its application to investigate the properties and regulation of gap junction channels and hemichannels.

Intercellular communication is essential for the coordination of physiological processes between cells in a variety of organs and tissues, including the ...

In Vivo Microinjection and Electroporation of Mouse Testis

This video and article contribution gives a comprehensive description of microinjection and electroporation of mouse testis in vivo. This particular transfection technique for testicular mouse cells allows the study of unique processes in spermatogenesis.
The following protocol focuses on transfection of testicular mouse cells with plasmid constructs. Specifically, we used the reporter vector pEGFP-C1, which expresses enhanced green fluorescent protein (eGFP) and also the pDsRed2-N1 vector expressing red fluorescent protein (DsRed2). Both encoded reporter genes were under the control of the human cytomegalovirus immediate-early promoter (CMV).
For performing gene transfer into mouse testes, the reporter plasmid constructs are injected into testes of living mice. To that end, the testis of an anaesthetized animal is exposed and the site of microinjection is prepared. Our preferred place of injection is the efferent duct, with the ultimately connected rete testis as the anatomical transport route of the spermatozoa between the testis and the epididymis. In this way, the filling of the seminiferous tubules after microinjection is excellently managed and controlled due to the use of stained DNA solutions. After observing a sufficient filling of the testis by its colored tubule structure, the organ is electroporated. This enables the transfer of the DNA solution into the testicular&#160;cells. Following 3 days of incubation, the testis is removed and investigated under the microscope for green or red&#160;fluorescence, illustrating transfection success.
Generally, this protocol can be employed for delivering DNA- or RNA- constructs into living mouse testis in order to (over)express or knock down genes, facilitating in vivo gene function analysis. Furthermore, it is suitable for studying reporter constructs or putative gene regulatory elements. Thus, the main advantages of the electroporation technique are fast performance in combination with low effort as well as the moderate technical equipment and skills required compared to alternative techniques.

In Vivo Microinjection and Electroporation of Mouse Testis

This article describes microinjection and electroporation of mouse testis in&#160;vivo as a&#160;transfection technique for testicular mouse cells to study unique processes of spermatogenesis. The presented protocol involves steps of glass capillary preparation, microinjection via the efferent duct, and transfection by electroporation.

This video and article contribution gives a comprehensive description of microinjection and electroporation of mouse testis in vivo. This particular ...

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a versatile analytical tool for characterizing viscoelastic mechanical properties for a variety of materials ranging from hard (plastic, glass, metal, etc.) surfaces to cells on any substrate. It has been widely used to measure the stiffness of cells, but less frequently used to measure the stiffness of aortas. In this paper, we will describe the procedures for using AFM in contact mode to measure the ex vivo elastic modulus of unloaded mouse arteries. We describe our procedure for isolation of mouse aortas, and then provide detailed information for the AFM analysis. This includes step-by-step instructions for alignment of the laser beam, calibration of the spring constant and deflection sensitivity of the AFM probe, and acquisition of force curves. We also provide a detailed protocol for data analysis of the force curves.

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

We present detailed protocols for isolation of aortas from mouse and measurement of their elastic modulus using atomic force microscopy.

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a ...

Preparation and Observation of Thick Biological Samples by Scanning Transmission Electron Tomography

This report describes a protocol for preparing thick biological specimens for further observation using a scanning transmission electron microscope. It also describes an imaging method for studying the 3D structure of thick biological specimens by scanning transmission electron tomography. The sample preparation protocol is based on conventional methods in which the sample is fixed using chemical agents, treated with a heavy atom salt contrasting agent, dehydrated in a series of ethanol baths, and embedded in resin. The specific imaging conditions for observing thick samples by scanning transmission electron microscopy are then described. Sections of the sample are observed using a through-focus method involving the collection of several images at various focal planes. This enables the recovery of in-focus information at various heights throughout the sample. This particular collection pattern is performed at each tilt angle during tomography data collection. A single image is then generated, merging the in-focus information from all the different focal planes. A classic tilt-series dataset is then generated. The advantage of the method is that the tilt-series alignment and reconstruction can be performed using standard tools. The collection of through-focal images allows the reconstruction of a 3D volume that contains all of the structural details of the sample in focus.

This report describes a sample preparation protocol and specific imaging conditions for performing scanning transmission electron tomography of thick biological specimens.

This report describes a protocol for preparing thick biological specimens for further observation using a scanning transmission electron microscope. It ...