Name: nucleofection and in vivo propagation of chicken eimeria parasites
Uploaded: 2020-02-14T12:00:00.000+00:00
Duration: 8 min 26 s
Description: Watch this Scientific Journal Video about Nucleofection and In Vivo Propagation of Chicken Eimeria Parasites at JoVE.com

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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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 vein resolved the the inability of sporozoites of E. acervulina to be inoculated via the cloacal route (Zhang et al., unpublished data). Here, we described a detailed transfection procedure to help researchers nucleofect eimerian parasites.
Previous studies showed that it was feasible to inject transfected sporozoites into the intestinal lumen of rabbits in a laparotomy for in vivo stable transfection of E. magna16 and E. intestinalis17. According to our experience, there was higher efficiency when sorting sporocysts by FACS instead of oocysts. There were also reports about transfection of unsporulated oocysts of E. maxima using a gene gun system or successful electroporation of sporulated oocysts with eGFP-Ham-OTU RNA18,19. Thus, our future studies explore transfection of oocysts or sporocysts to simplify the transfection procedures in Eimeria parasites.
The transfection success in eimerian parasites could enable genetically modified Eimeria to be used as vaccine vehicles to carry heterologous antigens, such as CjaA from C. jejuni13. Although transfection efficiency in Eimeria has been significantly improved, gene editing technology continues to have limitations in eimerian parasites. With the development of transfection in Eimeria, CRISPR/CAS9 technology in Eimeria (Hu et al., unpublished data) could lead to genetic manipulation of Eimeria.
In conclusion, this protocol provides a detailed procedure for nucleofection in chicken Eimeria. The transfection of sporozoites or merozoites is valuable for the study of gene function in Eimeria.

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><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 density gradient solutions. The oocysts of E. necatrix (Figure 3A) and E. tenella (Figure 3B) after nucleofecting the merozoites or sporozoites were also shown. The transfection efficiency of first-generation oocysts after nucleofecting the sporozoites is about 3-10%, in general. However, after nucleofecting the merozoites, the transfection efficiency of second-generation oocysts is only a few thousandths (Transfection efficiency of first-generation oocysts could not be calculated).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60552/60552fig1.jpg" /> 
Figure 1: Purification of sporozoites. (A) Purify sporozoites through the density-gradient centrifugation with density gradient solutions. (B) Purify sporozoites through the DE-52-cellulose column. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/60552/60552fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60552/60552fig2.jpg" /> 
Figure 2: The 2nd generation meronts and merozoites of E. necatrix along with the sporocysts and sporozoites of E. tenella. (A) Mature 2nd generation meronts of E. necatrix. (B) The released 2nd generation merozoites of E. necatrix. (C) Sporocysts of E. tenella after purification. (D) Sporozoites E. tenella after purification. The scale bar is 10 &#181;m. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/60552/60552fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60552/60552fig3.jpg" /> 
Figure 3: The oocysts obtained after infecting with the nucleofected merozoites or sporozoites. (A) The oocysts of E. necatrix after infecting with the nucleofected merozoites. (B) The oocysts of E. tenella after infecting with nucleofected sporozoites. The scale bar is 10 &#181;m. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/60552/60552fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Buffer</td>
			<td>Composition</td>
		</tr>
		<tr>
			<td>Digestion buffer</td>
			<td>0.25 g trypsin, 0.5 g sodium taurodeoxycholate hydrate, 100 ml PBS or HBSS</td>
		</tr>
		<tr>
			<td>0.1 M NaOH</td>
			<td>2 g NaOH, 500 ml water</td>
		</tr>
		<tr>
			<td>0.1 M HCl</td>
			<td>4.3 ml concentrated hydrochloric acid, 500 ml water</td>
		</tr>
		<tr>
			<td>Glycine eluent buffer</td>
			<td>0.75 g glycine, 7.9 g NaCl, 500 ml water</td>
		</tr>
		<tr>
			<td>Excystation buffer</td>
			<td>0.75% trypsin, 10% chicken bile, PBS</td>
		</tr>
		<tr>
			<td>1 &#215; DG gradient stock solution(Percoll)</td>
			<td>90% 1 &#215; DG gradient stock solution(Percoll), 10% PBS (10 X)</td>
		</tr>
		<tr>
			<td>Nucleofection buffer I</td>
			<td>ATP-disodium 2 g, MgCl2-6H2O 1.2 g, 10 ml water</td>
		</tr>
		<tr>
			<td>Nucleofection buffer II</td>
			<td>KH2PO4 6 g, NaHCO3 0.6 g, glucose 0.2 g, 500 ml water</td>
		</tr>
		<tr>
			<td colspan="2">*water: distilled water</td>
		</tr>
	</tbody>
</table>
Table 1: Composition of Buffers.

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 ...

nucleofection-and-in-vivo-propagation-of-chicken-eimeria-parasites

Universidad San Sebastian

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

Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach

The cone photoreceptor-enriched cultures derived from embryonic chick retinas have become an indispensable tool for researchers around the world studying the biology of retinal neurons, particularly photoreceptors. The applications of this system go beyond basic research, as they can easily be adapted to high throughput technologies for drug development. However, genetic manipulation of retinal photoreceptors in these cultures has proven to be very challenging, posing an important limitation to the usefulness of the system. We have recently developed and validated an ex&#160;ovo plasmid electroporation technique that increases the rate of transfection of retinal cells in these cultures by five-fold compared to other currently available protocols1. In this method embryonic chick eyes are enucleated at stage 27, the RPE is removed, and the retinal cup is placed in a plasmid-containing solution and electroporated using easily constructed custom-made electrodes. The retinas are then dissociated and cultured using standard procedures. This technique can be applied to overexpression studies as well as to the downregulation of gene expression, for example via the use of plasmid-driven RNAi technology, commonly achieving transgene expression in 25% of the photoreceptor population. The video format of the present publication will make this technology easily accessible to researchers in the field, enabling the study of gene function in primary retinal cultures. We have also included detailed explanations of the critical steps of this procedure for a successful outcome and reproducibility.

Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach

Embryonic chick retinal cell cultures constitute valuable tools for the study of photoreceptor biology. We have developed an efficient gene transfer technique based on ex&#160;ovo plasmid electroporation of the retinas prior to culture. This technique considerably increases transfection efficiencies over currently available protocols, making genetic manipulation feasible for this system.

The cone photoreceptor-enriched cultures derived from embryonic chick retinas have become an indispensable tool for researchers around the world studying ...

Developmental Biology

Gene Transfer into the Chicken Auditory Organ by In Ovo Micro-electroporation

Chicken embryos are ideal model systems for studying embryonic development as manipulations of gene function can be conducted with relative ease in ovo. The inner ear auditory sensory organ is critical for our ability to hear. It houses a highly specialized sensory epithelium that consists of mechano-transducing hair cells (HCs) and surrounding glial-like supporting cells (SCs). Despite structural differences in the auditory organs, molecular mechanisms regulating the development of the auditory organ are evolutionarily conserved between mammals and aves. In ovo electroporation is largely limited to early stages at E1 - E3. Due to the relative late development of the auditory organ at E5, manipulations of the auditory organ by in ovo electroporation past E3 are difficult due to the advanced development of the chicken embryo at later stages. The method presented here is a transient gene transfer method for targeting genes of interest at stage E4 - E4.5 in the developing chicken auditory sensory organ via in ovo micro-electroporation. This method is applicable for gain- and loss-of-functions with conventional plasmid DNA-based expression vectors and can be combined with in ovo cell proliferation assay by adding EdU (5-ethynyl-2&#180;-deoxyuridine) to the whole embryo at the time of electroporation. The use of green or red fluorescent protein (GFP or RFP) expression plasmids allows the experimenter to quickly determine whether the electroporation successfully targeted the auditory portion of the developing inner ear. In this method paper, representative examples of GFP electroporated specimens are illustrated; embryos were harvested 18 - 96 hr&#160;after electroporation and targeting of GFP to the pro-sensory area of the auditory organ was confirmed by RNA in situ hybridization. The method paper also provides an optimized protocol for the use of the thymidine analog EdU to analyze cell proliferation; an example of an EdU based cell proliferation assay that combines immuno-labeling and click EdU chemistry is provided.

Gene Transfer into the Chicken Auditory Organ by In Ovo Micro-electroporation

The auditory organ mediates hearing. Here we present a modified in ovo micro-electroporation method optimized for studying auditory progenitor cell proliferation and differentiation in the developing chicken auditory organ.

Chicken embryos are ideal model systems for studying embryonic development as manipulations of gene function can be conducted with relative ease in ovo. ...

In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

Dopaminergic neurons located in the ventral midbrain control movement, emotional behavior, and reward mechanisms1-3. The dysfunction of ventral midbrain dopaminergic neurons is implicated in Parkinson's disease, Schizophrenia, depression, and dementia1-5. Thus, studying the regulation of midbrain dopaminergic neuron differentiation could not only provide important insight into mechanisms regulating midbrain development and neural progenitor fate specification, but also help develop new therapeutic strategies for treating a variety of human neurological disorders.
Dopaminergic neurons differentiate from neural progenitors lining the ventricular zone of embryonic ventral midbrain. The development of neural progenitors is controlled by gene expression programs6,7. Here we report techniques utilizing electroporation to express genes specifically in the midbrain of Hamburger Hamilton (HH) stage 11 (thirteen somites, 42 hours) chick embryos8,9. The external development of chick embryos allows for convenient experimental manipulations at specific embryonic stages, with the effects determined at later developmental time points10-13. Chick embryonic neural tubes earlier than HH stage 13 (nineteen somites, 48 hours) consist of multipotent neural progenitors that are capable of differentiating into distinct cell types of the nervous system. The pCAG vector, which contains both a CMV promoter and a chick &beta;-actin enhancer, allows for robust expression of Flag or other epitope-tagged constructs in embryonic chick neural tubes14. In this report, we emphasize special measures to achieve regionally restricted gene expression in embryonic midbrain dopaminergic neuron progenitors, including how to inject DNA constructs specifically into the embryonic midbrain region and how to pinpoint electroporation with small custom-made electrodes. Analyzing chick midbrain at later stages provides an excellent in vivo system for plasmid vector-mediated gain-of-function and loss-of-function studies of midbrain development. Modification of the experimental system may extend the assay to other parts of the nervous system for performing fate mapping analysis and for investigating the regulation of gene expression.

In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

To assess the function and the regulation of genes during the development of midbrain dopaminergic neurons, we describe a method that involves in ovo electroporation of plasmid DNA constructs into embryonic chick ventral midbrain dopaminergic neuron progenitors. This technique can be used to achieve efficient expression of genes of interest to study different aspects of midbrain development and dopaminergic neuron differentiation.

Dopaminergic neurons located in the ventral midbrain control movement, emotional behavior, and reward mechanisms1-3. The dysfunction of ventral midbrain ...

Neuroscience

Influenza Virus Propagation in Embryonated Chicken Eggs

Influenza infection is associated with about 36,000 deaths and more than 200,000 hospitalizations every year in the United States. The continuous emergence of new influenza virus strains due to mutation and re-assortment complicates the control of the virus and necessitates the permanent development of novel drugs and vaccines. The laboratory-based study of influenza requires a reliable and cost-effective method for the propagation of the virus. Here, a comprehensive protocol is provided for influenza A virus propagation in fertile chicken eggs, which consistently yields high titer viral stocks. In brief, serum pathogen-free (SPF) fertilized chicken eggs are incubated at 37 &#176;C and 55-60% humidity for 10 &#8211; 11 days. Over this period, embryo development can be easily monitored using an egg candler. Virus inoculation is carried out by injection of virus stock into the allantoic cavity using a needle. After 2 days of incubation at 37 &#176;C, the eggs are chilled for at least 4 hr at 4 &#176;C. The eggshell above the air sac and the chorioallantoic membrane are then carefully opened, and the allantoic fluid containing the virus is harvested. The fluid is cleared from debris by centrifugation, aliquoted and transferred to -80 &#176;C for long-term storage. The large amount (5-10 ml of virus-containing fluid per egg) and high virus titer which is usually achieved with this protocol has made the usage of eggs for virus preparation our favorable method, in particular for in vitro studies which require large quantities of virus in which high dosages of the same virus stock are needed.

Fertile chicken eggs are widely used to produce large amounts of human influenza A virus as they provide a convenient and cost-effective system to prove high yields of virus.

Influenza infection is associated with about 36,000 deaths and more than 200,000 hospitalizations every year in the United States. The continuous ...

Immunology and Infection

Loop-mediated Isothermal Amplification (LAMP) Assays for the Species-specific Detection of Eimeria that Infect Chickens

Eimeria species parasites, protozoa which cause the enteric disease coccidiosis, pose a serious threat to the production and welfare of chickens. In the absence of effective control clinical coccidiosis can be devastating. Resistance to the chemoprophylactics frequently used to control Eimeria is common and sub-clinical infection is widespread, influencing feed conversion ratios and susceptibility to other pathogens such as Clostridium perfringens. Despite the availability of polymerase chain reaction (PCR)-based tools, diagnosis of Eimeria infection still relies almost entirely on traditional approaches such as lesion scoring and oocyst morphology, but neither is straightforward. Limitations of the existing molecular tools include the requirement for specialist equipment and difficulties accessing DNA as template. In response a simple field DNA preparation protocol and a panel of species-specific loop-mediated isothermal amplification (LAMP) assays have been developed for the seven Eimeria recognised to infect the chicken. We now provide a detailed protocol describing the preparation of genomic DNA from intestinal tissue collected post-mortem, followed by setup and readout of the LAMP assays. Eimeria species-specific LAMP can be used to monitor parasite occurrence, assessing the efficacy of a farm&#8217;s anticoccidial strategy, and to diagnose sub-clinical infection or clinical disease with particular value when expert surveillance is unavailable.

Loop-mediated Isothermal Amplification LAMP Assays for the Species-specific Detection of Eimeria that Infect Chickens

Diagnosis of Eimeria infection in chickens remains demanding. Parasite morphology- and host pathology-led approaches are commonly inconclusive, while molecular approaches based on PCR have proven demanding in cost and expertise. The aim of this protocol is to establish loop-mediated isothermal amplification (LAMP) as a straightforward molecular diagnostic for eimerian infection.

Eimeria species parasites, protozoa which cause the enteric disease coccidiosis, pose a serious threat to the production and welfare of chickens. In the ...

Single Cell Transfection in Chick Embryos

A central theme in developmental biology is the diversification of lineages and the elucidation of underlying molecular mechanisms. This entails a thorough analysis of the fates of single cells under normal and experimental conditions. To this end, transfection methods that target single progenitors are a prerequisite. We describe here a technically straightforward method for transfecting single cells in chicken tissues in-ovo, allowing reliable lineage tracing as well as genetic manipulation. Specific tissue domains are targeted within the somite or neural tube, and DNA is injected directly into the epithelium of interest, resulting in sporadic transfection of single cells. Using reporters, clonal populations may consequently be traced for up to three days, and behavior of genetically manipulated clonal populations can be compared with that of controls. This method takes advantage of the accessibility of the chick embryo along with emerging tools for genetic manipulation. We compare and discuss its advantages over the widely-used electroporation method, and possible applications and use in additional in-vivo models are also suggested. We advocate the use of this method as a significant addition and complement for existing lineage tracing and genetic interference tools.

Using fine tip micropipettes we inject plasmid DNA into subdomains of chicken somites or neural tubes. The concentration of the plasmid is adjusted to generate single transfected cells. We then allow the cells to develop into clonal populations.

A central theme in developmental biology is the diversification of lineages and the elucidation of underlying molecular mechanisms. This entails a ...

In Ovo Intravascular Injection in Chicken Embryos

As a classical model system of embryo biology, the chicken embryo has been used to investigate embryonic development and differentiation. Delivering exogenous materials into chicken embryos has a great advantage for studying gene function, transgenic breeding, and chimera preparation during embryonic development. Here we show the method of in ovo intravascular injection whereby exogenous materials such as plasmid vectors or modified primordial germ cells (PGCs) can be transferred into donor chicken embryos at early developmental stages. The results show that the intravascular injection through the dorsal aorta and head allows injected materials to diffuse into the whole embryo through the blood circulatory system. In the presented protocol, the efficacy of exogenous plasmid and lentiviral vector introduction, and the colonization of injected exogenous PGCs in the recipient gonad, were determined by observing fluorescence in the embryos. This article describes detailed procedures of this method, thereby providing an excellent approach to studying gene function, embryo and developmental biology, and gonad-chimeric chicken production. In conclusion, this article will allow researchers to perform in ovo intravascular injection of exogenous materials into chicken embryos with great success and reproducibility.

In Ovo Intravascular Injection in Chicken Embryos

The overall goal of this paper is to describe how to perform in ovo intracellular injection of exogenous materials into chicken embryos. This approach is very useful to study the developmental biology of chicken embryos.

As a classical model system of embryo biology, the chicken embryo has been used to investigate embryonic development and differentiation. Delivering ...

Gene Transfer into Older Chicken Embryos by ex ovo Electroporation

The chicken embryo provides an excellent model system for studying gene function and regulation during embryonic development. In ovo electroporation is a powerful method to over-express exogenous genes or down-regulate endogenous genes in vivo in chicken embryos1. Different structures such as DNA plasmids encoding genes2-4, small interfering RNA (siRNA) plasmids5, small synthetic RNA oligos6, and morpholino antisense oligonucleotides7 can be easily transfected into chicken embryos by electroporation. However, the application of in ovo electroporation is limited to embryos at early incubation stages (younger than stage HH20 - according to Hamburg and Hamilton)8 and there are some disadvantages for its application in embryos at later stages (older than stage HH22 - approximately 3.5 days of development). For example, the vitelline membrane at later stages is usually stuck to the shall membrane and opening a window in the shell causes rupture of the vessels, resulting in death of the embryos; older embryos are covered by vitelline and allantoic vessels, where it is difficult to access and manipulate the embryos; older embryos move vigorously and is difficult to control the orientation through a relatively small window in the shell.
In this protocol we demonstrate an ex ovo electroporation method for gene transfer into chicken embryos at late stages (older than stage HH22). For ex ovo electroporation, embryos are cultured in Petri dishes9 and the vitelline and allantoic vessels are widely spread. Under these conditions, the older chicken embryos are easily accessed and manipulated. Therefore, this method overcomes the disadvantages of in ovo electroporation applied to the older chicken embryos. Using this method, plasmids can be easily transfected into different parts of the older chicken embryos10-12.

Gene Transfer into Older Chicken Embryos by ex ovo Electroporation

A method of gene transfer into chicken embryos at later incubation stages (older than Hamburger and Hamilton stage (HH) 22) is described. This method overcomes disadvantages of in ovo electroporation applied to older chicken embryos and is a useful technique to study gene function and regulation at older developmental stages.

The chicken embryo provides an  excellent model system for studying gene function and regulation during  embryonic development. In ovo electroporation is ...

A Rapid In Vivo Bioassay for Developmentally Active Enhancers

A potential regulatory sequence (PRS) can be identified by several approaches including conservation analysis and transcription factor-specific chromatin immunoprecipitation followed by next generation sequencing (TF ChIP-seq). TF ChIP-seq generates a large data set (TF-regulatome) that includes PRSs and background interactions. These approaches require secondary low throughput validation in model systems that replicate the temporal and spatial specificity of the PRS. 
In this report, a protocol is described to rapidly validate activity of PRSs in their developmental context. Following selection of candidate PRSs, the associated sequences are isolated, cloned into a GFP reporter construct, and then transfected into chick embryos. For PRSs that are anticipated to be functional during early development, from gastrulation through early organogenesis, whole embryo electroporation (EP) is recommended. With this technique transfection, incubation and monitoring occurs ex ovo during early gastrulation. The entire embryo is transfected and as structures form, they can be monitored live for PRS activity. This ex ovo approach supports development up to early limb bud outgrowth. 
If the functional activity of a PRS is expected to be later in development or if the investigation is to ascertain its activity during the development of a specific organ, targeted regional electroporation (TREP) is used. This approach requires injection of construct DNA directly into the developing organ, precursor or adjacent cavity prior to electroporation. An approach to transfect the presumptive limb bud using TREP is also outlined and illustrated. TREP supports a rapid organ-specific validation of a PRS' activity. Both techniques can also be used to investigate regulatory domains by site-directed mutagenesis of the PRS-reporter construct. 
The chick as a bioassay offers a rapid effective tool for PRS validation, functional domain determination, and further characterization of the regulatory events responsible for development.

With the increased use of transcription factor-specific ChIP-seq technology, techniques to validate potential regulatory regions are critical. The authors describe a rapid chick bioassay to validate the activity of regulatory sequences functioning during development.

A potential regulatory sequence (PRS) can be identified by several approaches including conservation analysis and transcription factor-specific chromatin ...

In ovo Electroporation of Chicken Embryos

Electroporation is a technique used in biomedical research that allows for the manipulation of gene expression via the delivery of foreign genetic material into cells. More specifically, in ovo electroporation is performed on early developing chicks (Gallus gallus domesticus) contained within their eggshells. In this procedure, DNA or knockdown constructs are first injected into a target tissue. However, the genetic material is unable to penetrate the plasma membrane to carry out its function within the cell. To solve this problem, an electrical field is applied, causing temporary disruptions to membrane stability. This electric field also causes the negatively charged nucleic acids to migrate toward the positively charged electrode through the holes in the plasma membrane, thus effectively driving the DNA or knockdown construct into the cell. The major advantage of this technique is that the delivery of genetic material can be localized to isolated cell types at specific developmental time points. As a result, the genetic mechanisms that govern individual developmental events can be examined.
This video provides an overview of the principles behind in ovo electroporation and introduces the tools required for the technique, including capillary needles, electrodes, and an electroporator. A step-by-step protocol for carrying out the procedure is also presented prior to discussion of a few fascinating examples of how the technique is used to perform a variety of genetic manipulations in chicken embryos.

Nucleofection and In Vivo Propagation of Chicken Eimeria Parasites

Summary

Explore More Videos

Chapters in this video

Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach

Gene Transfer into the Chicken Auditory Organ by In Ovo Micro-electroporation

In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

Influenza Virus Propagation in Embryonated Chicken Eggs

Loop-mediated Isothermal Amplification (LAMP) Assays for the Species-specific Detection of Eimeria that Infect Chickens

Single Cell Transfection in Chick Embryos

In Ovo Intravascular Injection in Chicken Embryos

Gene Transfer into Older Chicken Embryos by ex ovo Electroporation

A Rapid In Vivo Bioassay for Developmentally Active Enhancers

In ovo Electroporation of Chicken Embryos