Name: generating genetically modified plasmodium berghei sporozoites
Uploaded: 2023-05-05T12:50:00
Description: Watch this Scientific Journal Video about Generating Genetically Modified Plasmodium berghei Sporozoites at JoVE.com

This work was supported by the National Institutes of Health grant AI168307 to SPK.&#160; We thank the UGA CTEGD Flow Cytometry Core and the UGA CTEGD Microscopy Core. We also acknowledge the contributions of Ash Pathak, Anne Elliot, and the staff of UGA Sporocore in optimizing the protocol. We want to thank Dr. Daichi Kamiyama for valuable insights, discussion, and the parent plasmids containing GFP11 and GFP1-10. We would also like to thank members of the Kurup lab for their constant support, patience, and encouragement.

All research involving vertebrate animals in our laboratory was performed in compliance with the University of Georgia animal use guidelines and protocols.
1. Generation of P. berghei -infected mice
<ol>
	<li>Initiate blood-stage infection in male or female, 6-8-week-old C57BL/6 (B6) mice using wild-type P. berghei parasites. To do this, transfer cryopreserved P. berghei-infected blood (2 x 105 infected RBCs), reconstituted in 400 &#181;...

<ol>
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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=A+comprehensive+evaluation+of+rodent+malaria+parasite+genomes+and+gene+expression.">A comprehensive evaluation of rodent malaria parasite genomes and gene expression.</a> BMC Biology. 12, 86 (2014).</li><li>Janse, C. J., Ramesar, J., Waters, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-efficiency+transfection+and+drug+selection+of+genetically+transformed+blood+stages+of+the+rodent+malaria+parasite+Plasmodium+berghei.">High-efficiency transfection and drug selection of genetically transformed blood stages of the rodent malaria parasite Plasmodium berghei.</a> Nature Protocols. 1 (1), 346-356 (2006).</li><li>Tripathi, A. 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We have used the above protocol in our laboratory to create several lines of transgenic P. berghei parasites. Though optimized for P. berghei, we have also successfully used this protocol to generate transgenic P. yoelii sporozoites. After injecting the transfected schizonts into mice, parasites are detectable typically no later than 3 d.p.i. in all groups, including the no plasmid control. Selection is started only once parasitemia has been detected to ensure the viability of parasites followi...

Despite advances in drug development and research into malaria prevention and treatment, the global disease burden of malaria remains high. Over half a million people die of malaria each year, with the highest levels of mortality seen among children living in malaria-endemic regions, such as sub-Saharan Africa1. Malaria is caused by the parasite Plasmodium, which is transmitted to humans through the bite of female Anopheles mosquitoes bearing the parasite in their salivary glands. The infectious stage of Plasmodium-the sporozoites-are deposited in the skin of the vertebrate hosts during a blood meal and travel through the bloodstream to infect liver cells, where they undergo mandatory development (constituting pre-erythrocytic malaria) prior to infecting the erythrocytes. The infection of the erythrocytes initiates the blood-stage of malaria and is responsible for the entirety of the morbidity and mortality associated with the disease2,3.
The obligate nature of the pre-erythrocytic development of Plasmodium has made it an attractive target for prophylactic vaccine and drug development efforts4. A prerequisite for studying the biology of pre-erythrocytic malaria, as well as the development of vaccines or drugs targeting the liver stage, is access to Plasmodium sporozoites. Furthermore, our ability to generate genetically modified Plasmodium sporozoites has been instrumental in the success of such research endeavors5,6,7,8,9. Transgenic Plasmodium lines expressing fluorescent or luminescent reporter proteins have allowed us to track their development in vivo and in vitro10,11. Genetically attenuated parasites (GAPs), generated through the deletion of multiple genes in Plasmodium, are also some of the most promising vaccine candidates12,13.
Rodent and non-human primate malaria models have helped us understand the mechanisms of host-parasite interactions in human malaria due to the similarities in biology and life cycle among Plasmodium species14. The use of Plasmodium species that infect rodents, but not humans (e.g., P. berghei) allows the maintenance of the complete parasite life cycle and the generation of infectious sporozoites for studying liver-stage malaria in a controlled, biosafety level 1 setting. A variety of separate protocols already exist for the generation of transgenic blood-stage Plasmodium parasites15, infection of mosquitoes16, and isolation of sporozoites17. Here, we outline a comprehensive protocol combining these methodologies in order to generate and isolate transgenic P. berghei sporozoites, utilizing the novel transgenic strain PbGFP11 as an example.&#160;PbGFP11 traffics the 11th &#946;-strand of super-folder green fluorescent protein (GFP), GFP11,&#160;into the parasitophorous vacuole (PV) generated in the host hepatocytes. PbGFP11&#160;is used in conjunction with transgenic hepatocytes (Hepa1-6 background) expressing residues that constitute the GFP 1-10 fragment (GFP1-10) in the cytoplasm (Hepa GFP1-10 cells). PbGFP11 reports PV lysis in the host hepatocytes through self-complementation and the reformation of functional GFP and the green fluorescence signal18. Of note, GFP11 is encoded as a series of seven tandem sequences in PbGFP11&#160;to enhance the resulting fluorescence signal. Upon staining PbGFP11&#160;sporozoites with the cytoplasmic dye CellTrace Violet (CTV), we can track the parasites. The lysis of such CTV-stained intracellular parasites itself results in leakage of CTV into the host cell cytoplasm and staining of the host cell. In addition to visualizing and distinguishing the lysis of Plasmodium PV and/or the parasite in host hepatocytes, this system can be reliably used to study the immune pathways responsible for either of these processes, through the genetic or therapeutic perturbation of the molecular components of such pathways.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>30 G x 1/2&#34; Syringe needle</td>
			<td>Exel international</td>
			<td>26437</td>
			<td></td>
		</tr>
		<tr>
			<td>Alsever&#39;s solution</td>
			<td>Sigma-Aldritch</td>
			<td>A3551-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Amaxa Basic Parasite Nucleofector Kit 2</td>
			<td>Lonza</td>
			<td>VMI-1021</td>
			<td></td>
		</tr>
		<tr>
			<td>Avertin (2,2,2-Tribromoethanol)</td>
			<td>TCI America</td>
			<td>T1420</td>
			<td></td>
		</tr>
		<tr>
			<td>Blood collection tubes</td>
			<td>BD bioscience</td>
			<td>365967</td>
			<td>for serum collection</td>
		</tr>
		<tr>
			<td>C-Chip disposable hematocytometer</td>
			<td>INCYTO</td>
			<td>DHC-N01-5</td>
			<td></td>
		</tr>
		<tr>
			<td>CellVeiw Cell Culture Dish</td>
			<td>Greiner Bio-One</td>
			<td>627860</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge 5425</td>
			<td>Eppendorf</td>
			<td>5405000107</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge 5910R</td>
			<td>Eppendorf</td>
			<td>5910R</td>
			<td>For gradient centrifugation</td>
		</tr>
		<tr>
			<td>Delta Vision II - Inverted microscope system</td>
			<td>Olympus</td>
			<td>IX-71</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl Sulfoxide</td>
			<td>Sigma</td>
			<td>D5879-500ml</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>GenClone</td>
			<td>25-525</td>
			<td></td>
		</tr>
		<tr>
			<td>GFP11 plasmid</td>
			<td>Kurup Lab</td>
			<td>pSKspGFP11</td>
			<td>Generated from PL0017 plasmid</td>
		</tr>
		<tr>
			<td>Giemsa Stain</td>
			<td>Sigma-Aldritch</td>
			<td>48900-1L-F</td>
			<td></td>
		</tr>
		<tr>
			<td>Hepa GFP1-10 cells</td>
			<td>Kurup Lab</td>
			<td>Hepa GFP1-10</td>
			<td>Generated from Hepa 1-6 cells (ATCC Cat# CRL-1830)</td>
		</tr>
		<tr>
			<td>Mouse Serum</td>
			<td></td>
			<td></td>
			<td>Used for mosquito dissection media</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Millipore-Sigma</td>
			<td>SX0420-5</td>
			<td>1.5 M and 0.15 M for percoll solution</td>
		</tr>
		<tr>
			<td>Nucleofector II</td>
			<td>Amaxa Biosystems (Lonza)</td>
			<td></td>
			<td>Program U-033 used for RBC electroporation</td>
		</tr>
		<tr>
			<td>Pasteur pipette</td>
			<td>VWR</td>
			<td>14673-043</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Sigma-Aldritch</td>
			<td>P0781-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Percoll (Density gradient stock medium)</td>
			<td>Cytivia</td>
			<td>17-0891-02</td>
			<td>Details in protocol</td>
		</tr>
		<tr>
			<td>PL0017 Plasmid</td>
			<td>BEI Resources</td>
			<td>MRA-786</td>
			<td></td>
		</tr>
		<tr>
			<td>Pyrimethamine (for oral administration)</td>
			<td>Sigma</td>
			<td>46706</td>
			<td>Preparation details: Add 17.5 mg Pyrimethamine to 2.5 mL of DMSO. Vortex, if needed to dissolve completely; Adjust pH of 225 mL of dH2O to 4 using HCL. Add Pyrimethamine in DMSO to water and bring to 250 mL. Add 10 g of sugar to encourage regular consumption of drugged water. Pyrimethamine is light sensitive. Use dark bottle or aluminum foil covered bottle when treating mice.</td>
		</tr>
		<tr>
			<td>RPMI 1640</td>
			<td>Corning</td>
			<td>15-040-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>SoftWoRx microscopy software</td>
			<td>Applied Precision</td>
			<td>v6.1.3</td>
			<td></td>
		</tr>
	</tbody>
</table>

generating genetically modified plasmodium berghei sporozoites

Malaria is a deadly disease caused by the parasite Plasmodium and is transmitted through the bite of female Anopheles mosquitoes. The sporozoite stage of Plasmodium deposited by mosquitoes in the skin of vertebrate hosts undergoes a phase of mandatory development in the liver before initiating clinical malaria. We know little about the biology of Plasmodium development in the liver; access to the sporozoite stage and the ability to genetically modify such sporozoites are critical tools for studying the nature of Plasmodium infection and the resulting immune response in the liver. Here, we present a comprehensive protocol for the generation of transgenic Plasmodium berghei sporozoites. We genetically modify blood-stage P. berghei and use this form to infect Anopheles mosquitoes when they take a blood meal. After the transgenic parasites undergo development in the mosquitoes, we isolate the sporozoite stage of the parasite from the mosquito salivary glands for in vivo and in vitro experimentation. We demonstrate the validity of the protocol by generating sporozoites of a novel strain of P. berghei expressing the green fluorescent protein (GFP) subunit 11 (GFP11), and show how it could be used to investigate the biology of liver-stage malaria.

Determining the frequency and development of schizonts is critical for assuring that enough viable parasites are in the optimal stage for transfection. Immature schizonts can be differentiated from fully mature schizonts by the presence of fewer merozoites that do not fill the entire intracellular space of the RBC (Figure 1B). It is important to note that when making blood smears from cultured blood, infected RBCs may break open, resulting in the observation of free, extracellular merozoites...

Watch this Scientific Journal Video about Generating Genetically Modified Plasmodium berghei Sporozoites at JoVE.com

Generating Genetically Modified Plasmodium berghei Sporozoites

Malaria is transmitted through inoculation of the sporozoite stage of Plasmodium by infected mosquitoes. Transgenic Plasmodium has allowed us to understand the biology of malaria better and has contributed directly to malaria vaccine development efforts. Here, we describe a streamlined methodology to generate transgenic Plasmodium berghei sporozoites.

Generating Genetically Modified Plasmodium berghei Sporozoites

Malaria is a deadly disease caused by the parasite Plasmodium and is transmitted through the bite of female Anopheles mosquitoes. The sporozoite stage of ...

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