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>
	<li>WHO. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Geneva.">Geneva.</a> World Health Organization. , 1 (2020).</li><li>Cowman, A. F., Healer, J., Marapana, D., Marsh, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Malaria:+biology+and+disease.">Malaria: biology and disease.</a> Cell. 167 (3), 610-624 (2016).</li><li>Crompton, P. 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=Malaria+immunity+in+man+and+mosquito:+insights+into+unsolved+mysteries+of+a+deadly+infectious+disease.">Malaria immunity in man and mosquito: insights into unsolved mysteries of a deadly infectious disease.</a> Annual Review of Immunology. 32, 157-187 (2014).</li><li>Marques-da-Silva, C., Peissig, K., Kurup, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pre-erythrocytic+vaccines+against+malaria.">Pre-erythrocytic vaccines against malaria.</a> Vaccines. 8 (3), 400 (2020).</li><li>Balu, B., Adams, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advancements+in+transfection+technologies+for+Plasmodium.">Advancements in transfection technologies for Plasmodium.</a> International Journal for Parasitology. 37 (1), 1-10 (2007).</li><li>Rodriguez, A., Tarleton, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenic+parasites+accelerate+drug+discovery.">Transgenic parasites accelerate drug discovery.</a> Trends in Parasitology. 28 (3), 90-92 (2012).</li><li>Voorberg-vander Wel, A. 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=A+dual+fluorescent+Plasmodium+cynomolgi+reporter+line+reveals+in+vitro+malaria+hypnozoite+reactivation.">A dual fluorescent Plasmodium cynomolgi reporter line reveals in vitro malaria hypnozoite reactivation.</a> Communications Biology. 3, 7 (2020).</li><li>Christian, D. A., 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=Use+of+transgenic+parasites+and+host+reporters+to+dissect+events+that+promote+interleukin-12+production+during+toxoplasmosis.">Use of transgenic parasites and host reporters to dissect events that promote interleukin-12 production during toxoplasmosis.</a> Infection and Immunity. 82 (10), 4056-4067 (2014).</li><li>Montagna, G. N., 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=Antigen+export+during+liver+infection+of+the+malaria+parasite+augments+protective+immunity.">Antigen export during liver infection of the malaria parasite augments protective immunity.</a> mBio. 5 (4), e01321 (2014).</li><li>Amino, R., Menard, R., Frischknecht, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+of+malaria+parasites-recent+advances+and+future+directions.">In vivo imaging of malaria parasites-recent advances and future directions.</a> Current Opinion in Microbiology. 8 (4), 407-414 (2005).</li><li>Siciliano, G., Alano, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enlightening+the+malaria+parasite+life+cycle:+bioluminescent+Plasmodium+in+fundamental+and+applied+research.">Enlightening the malaria parasite life cycle: bioluminescent Plasmodium in fundamental and applied research.</a> Frontiers in Microbiology. 6, 391 (2015).</li><li>Othman, A. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+transgenic+parasites+in+malaria+vaccine+research.">The use of transgenic parasites in malaria vaccine research.</a> Expert Review of Vaccines. 16 (7), 1-13 (2017).</li><li>Kreutzfeld, O., Muller, K., Matuschewski, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+of+genetically+arrested+parasites+(GAPs)+for+a+precision+malaria+vaccine.">Engineering of genetically arrested parasites (GAPs) for a precision malaria vaccine.</a> Frontiers in Cellular and Infection Microbiology. 7, 198 (2017).</li><li>Otto, T. 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. K., Mlambo, G., Kanatani, S., Sinnis, P., Dimopoulos, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasmodium+falciparum+gametocyte+culture+and+mosquito+infection+through+artificial+membrane+feeding.">Plasmodium falciparum gametocyte culture and mosquito infection through artificial membrane feeding.</a> Journal of Visualized Experiments. (161), e61426 (2020).</li><li>Pacheco, N. D., Strome, C. P., Mitchell, F., Bawden, M. P., Beaudoin, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid,+large-scale+isolation+of+Plasmodium+berghei+sporozoites+from+infected+mosquitoes.">Rapid, large-scale isolation of Plasmodium berghei sporozoites from infected mosquitoes.</a> The Journal of Parasitology. 65 (3), 414-417 (1979).</li><li>Kamiyama, 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=Versatile+protein+tagging+in+cells+with+split+fluorescent+protein.">Versatile protein tagging in cells with split fluorescent protein.</a> Nature Communications. 7, 11046 (2016).</li><li>Bailey, J. W., 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=Guideline:+the+laboratory+diagnosis+of+malaria.+General+Haematology+Task+Force+of+the+British+Committee+for+Standards+in+Haematology.">Guideline: the laboratory diagnosis of malaria. General Haematology Task Force of the British Committee for Standards in Haematology.</a> British Journal of Haematology. 163 (5), 573-580 (2013).</li><li>Das, 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+systematic+literature+review+of+microscopy+methods+reported+in+malaria+clinical+trials.">A systematic literature review of microscopy methods reported in malaria clinical trials.</a> The American Journal of Tropical Medicine and Hygiene. 104 (3), 836-841 (2020).</li><li>de Oca, M. M., Engwerda, C., Haque, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasmodium+berghei+ANKA+(PbA)+infection+of+C57BL/6J+mice:+a+model+of+severe+malaria.">Plasmodium berghei ANKA (PbA) infection of C57BL/6J mice: a model of severe malaria.</a> Methods in Molecular Biology. 1031, 203-213 (2013).</li><li>Musiime, A. K., 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=Is+that+a+real+oocyst?+Insectary+establishment+and+identification+of+Plasmodium+falciparum+oocysts+in+midguts+of+Anopheles+mosquitoes+fed+on+infected+human+blood+in+Tororo,+Uganda.">Is that a real oocyst? Insectary establishment and identification of Plasmodium falciparum oocysts in midguts of Anopheles mosquitoes fed on infected human blood in Tororo, Uganda.</a> Malaria Journal. 18 (1), 287 (2019).</li><li>Marques-da-Silva, 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=Direct+type+I+interferon+signaling+in+hepatocytes+controls+malaria.">Direct type I interferon signaling in hepatocytes controls malaria.</a> Cell Reports. 40 (3), 111098 (2022).</li><li>Bowers, 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=Cryopreservation+of+Plasmodium+sporozoites.">Cryopreservation of Plasmodium sporozoites.</a> Pathogens. 11 (12), 1487 (2022).</li><li>Zander, R. A., 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=Th1-like+plasmodium-specific+memory+CD4++T+cells+support+humoral+immunity.">Th1-like plasmodium-specific memory CD4+ T cells support humoral immunity.</a> Cell Reports. 21 (7), 1839-1852 (2017).</li></ol>

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 ...

Currently we have a limited understanding of the biology of plasmodium infection of the liver. This protocol has helped us generate a lot of transgenic lines that has helped answer some critical questions about the biology of the organism. A fundamental understanding of the parasite and its biology are required to develop new tools and approaches to combat the infection of malaria.The method of transgenesis that we are describing here has helped us decipher some key and complex biology of the organism as well as the host. Demonstrating this procedure will be Carson Bowers, my laboratory manager. After generating P.Bergehei infected mice and euthanizing them, collect two milliliters of blood from two infected mice into five milliliters of Elsevier solution in a sterile environment.Centrifuge the blood at 450 G for eight minutes at room temperature. Discard the supernatant and re-suspend the cell pellet in 10 milliliters of complete RPMI media. Centrifuge the cell suspension again and re-suspend the pellet in 25 milliliters of complete RPMI media.Transfer the cell suspension to a T75 culture flask with a filter cap and incubate with gentle shaking to keep the cells in suspension. At the end of the incubation, collect 500 microliters of the sample. Centrifuge and re-suspend the pellet in 10 microliters of complete RPMI media.Next, prepare a thin blood smear. Stain the blood smear with Giemsa stain and determine the frequency of schizonts. For optimal transfection efficiency, 60 to 70%of the parasites should be in the schizont stage.Prepare a gradient centrifugation buffer by reconstituting 13 milliliters of density gradient stock medium in a 50 milliliter centrifugation tube. Transfer 25 milliliters of blood culture to a fresh 50 milliliter centrifugation tube. Then carefully layer 20 milliliters of gradient centrifugation buffer to the bottom of the centrifugation tube, using a narrow glass pipette to create a gradient column and ensure a clear division between the buffer and the media.Centrifuge the prepared buffer and media for 20 minutes at 450 G with no break at room temperature. Using a pasture pipette, carefully remove the schizont layer at the interface of the culture media and the gradient centrifugation buffer. Place it in a new 50 milliliter centrifugation tube.Re-suspend the isolated schizonts in complete RPMI media and increase the total volume to 40 milliliters. After centrifuging and discarding the supernatant, re-suspend the schizont in 10 milliliters of complete RPMI media. Then transfer one milliliter of this solution into a 1.5 milliliter micro centrifuge tube for each transfection and pellet the infected cells by centrifugation at 16, 000 G for five seconds.Next, combine 100 microliters of the electroporation buffer at room temperature with five micrograms of target plasmid DNA in a 1.5 milliliter micro centrifuge tube. After centrifuging the infected cells, remove the supernatant and carefully re-suspend the cells in the prepared nucleoefector solution plus plasmid DNA. Then transfer the suspension into electroporation, cuvettes, and transfect using a suitable nucleofector program.Next, add 100 microliters of complete RPMI media to the cuvette. Transfer the entire solution into a 1.5 milliliter micro centrifuge tube and place it on ice. Verify the parasitemia levels in the mice inoculated with the transfected schizont daily from two days post inoculation.Once the infection is confirmed, start drug selection by administering the drug orally and drinking water offered ad libitum. Monitor parasitemia using blood smears starting from six days after the initiation of pyromethymine treatment. When parasitemia reaches at least 1%transfer the parasites to naive B6 mice to create blood stage parasite stocks.Once parasitemia reaches 2%to 5%generate stocks and cryopreserve them. One day before mosquito infection, separate the female mosquitoes by placing a glove filled with water heated to 37 degrees Celsius on top of the cage containing both male and female mosquitoes. Use an insect aspirator to remove the female mosquitoes attracted to the heat source and transfer them to a smaller cage for infection.On the day of mosquito feeding and infection, determine parasitemia in the infected B6 mice. Parasitemia between 2%to 5%is optimal for mosquito infection. After verifying the parasitemia, transfer the infection to the female mosquitoes by placing the anesthetized mice on the caged female mosquitoes under sternal recumbency.Manually spread the front and back legs to provide the greatest surface area for mosquito feeding. Leave the infected mice in each cage for 15 minutes, checking every five minutes to ensure the mice are not awake. Once the feeding is complete and the mice are euthanized, dispose of them safely following the institution's guidelines.To verify successful infection within the mosquitoes, isolate the mosquitoes midguts by removing the terminal abdominal segment with forceps 10 days post-infection. Then view the midguts under polarized light to detect the presence of oocysts. At 18 days post-infection, dissect five to 10 mosquitoes to evaluate the number of sporozoites present.To isolate and count the sporozoites, carefully remove the head of the mosquito while applying pressure to the thorax with forceps or a fine dissecting needle. The salivary glands will remain attached to the removed head. Next, remove any additional mosquito debris from the salivary glands and place them in a micro centrifuge tube containing 400 microliters of mosquito dissection media.Then disrupt the isolated salivary glands by passing them through a 30 gauge needle syringe 15 to 20 times, Afterward, place 10 microliters of the disrupted salivary glands in mosquito dissection media into a hemocytometer and count. Finally, re-suspend the salivary glands in the desired concentration of mosquito dissection media or PBS for injection into mice or long-term cryo-preservation. Like microscopy images depicting mature and immature P.berghei schizont in parasitized mouse blood cultures are presented.Like microscopy images of a mosquito head with the salivary glands still intact during dissection, and the dissected salivary gland under lower and higher magnifications are shown. The generation of green fluorescence signal in the PB, GFP 11 infected HEPA GFP one to 10 host cells indicated the lysis of the parasitophorous vacuole. It's important to carefully remove the chaisson layer without disrupting the gradient interface.Also, it takes a bit of practice to reliably remove the salivary glands along with the head during spores light isolation. Following isolation, transgenic sporozoites can be cryo-preserved or used fresh for in vivo or in vitro infection studies.

generating-genetically-modified-plasmodium-berghei-sporozoites

Research

JoVE Journal

Immunology and Infection

1.0K vistas.  University of Georgia. Actualmente tenemos una comprensión limitada de la biología de la infección hepática por plasmodium. Este protocolo nos ha ayudado a generar una gran cantidad de líneas transgénicas que han ayudado a responder algunas preguntas críticas sobre la biología del organismo. Se requiere una comprensión fundamental del parásito y su biología para desarrollar nuevas herramientas y enfoques para combatir la infección de la malaria.El método de transgénesis que estamos describiendo...