We are grateful to Drusilla Stillinger, Kiona Parker, Parrish Powell and Madeline Nottoli for mosquito husbandry. Funding was provided by the University of California, Irvine Malaria Initiative. AAJ is a Donald Bren Professor at the University of California, Irvine.

1. Preparing mosquitoes for microinjection
<ol>
	<li>Seed a cage13 (~5000 cm3) with ~100 male and 200-300 female 1-2 day adult post-eclosion mosquitoes and allow them to mate for 2 days.</li>
	<li>After the mating period, provide mosquitoes a blood meal using either 2 mL of blood with an artificial feeding device or live anesthetized animals depending on insectary practices14. The following day provide mosquitoes a second blood meal to ensure that all mated fem...

<ol>
	<li>Feramisco, J., Perona, R., Lacal, J. C., Lacal, J. C., Feramisco, J., Perona, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Needle+Microinjection:+A+Brief+History.">Needle Microinjection: A Brief History.</a> Microinjection. Methods and Tools in Biosciences and Medicine. , (1999).</li><li>Windbichler, 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=A+synthetic+homing+endonuclease-based+gene+drive+system+in+the+human+malaria+mosquito.">A synthetic homing endonuclease-based gene drive system in the human malaria mosquito.</a> Nature. 473 (7346), 212-215 (2011).</li><li>Meredith, J. 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=Site-specific+integration+and+expression+of+an+anti-malarial+gene+in+transgenic+Anopheles+gambiae+significantly+reduces+Plasmodium+infections.">Site-specific integration and expression of an anti-malarial gene in transgenic Anopheles gambiae significantly reduces Plasmodium infections.</a> PLoS One. 6 (1), 14587 (2011).</li><li>Hammond, 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=CRISPR-Cas9+gene+drive+system+targeting+female+reproduction+in+the+malaria+mosquito+vector+Anopheles+gambiae.">CRISPR-Cas9 gene drive system targeting female reproduction in the malaria mosquito vector Anopheles gambiae.</a> Nature Biotechnology. 34 (1), 78-83 (2016).</li><li>Carballar-Lejarazu, R., 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=Next-generation+gene+drive+for+population+modification+of+the+malaria+vector+mosquito,+Anopheles+gambiae.">Next-generation gene drive for population modification of the malaria vector mosquito, Anopheles gambiae.</a> Proceedings of the National Academy of Sciences of the United States of America. 117 (37), 22805-22814 (2020).</li><li>Sim&#245;es, M. L., 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+Anopheles+FBN9+immune+factor+mediates+Plasmodium+species-specific+defense+through+transgenic+fat+body+expression.">The Anopheles FBN9 immune factor mediates Plasmodium species-specific defense through transgenic fat body expression.</a> Develomental &amp; Comparative Immunology. 67, 257-265 (2017).</li><li>Arik, A. J., 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=Increased+Akt+signaling+in+the+mosquito+fat+body+increases+adult+survivorship.">Increased Akt signaling in the mosquito fat body increases adult survivorship.</a> FASEB Journal. 4, 1404-1413 (2015).</li><li>Riabinina, O., 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=Organization+of+olfactory+centres+in+the+malaria+mosquito+Anopheles+gambiae.">Organization of olfactory centres in the malaria mosquito Anopheles gambiae.</a> Nature Communications. 7, 13010 (2016).</li><li>Kyrou, 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=A+CRISPR-Cas9+gene+drive+targeting+doublesex+causes+complete+population+suppression+in+caged+Anopheles+gambiae+mosquitoes.">A CRISPR-Cas9 gene drive targeting doublesex causes complete population suppression in caged Anopheles gambiae mosquitoes.</a> Nature Biotechnology. 36 (11), 1062-1066 (2018).</li><li>Dong, Y., Sim&#245;es, M. L., 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=Versatile+transgenic+multistage+effector-gene+combinations+for+Plasmodium+falciparum+suppression+in+Anopheles.">Versatile transgenic multistage effector-gene combinations for Plasmodium falciparum suppression in Anopheles.</a> Science Advances. 6 (20), (2020).</li><li>Grossman, G. L., 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=Germline+transformation+of+the+malaria+vector,+Anopheles+gambiae,+with+the+piggyBac+transposable+element.">Germline transformation of the malaria vector, Anopheles gambiae, with the piggyBac transposable element.</a> Insect Molecular Biology. 6, 597-604 (2001).</li><li>Benedict, M. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+in+Anopheles+research.+Chapter+3:+Specific+Anopheles+techniques.+3.1+Embryonic+Techniques.+3.1.1+Microinjection+methods+for+Anopheles+Embryos.">Methods in Anopheles research. Chapter 3: Specific Anopheles techniques. 3.1 Embryonic Techniques. 3.1.1 Microinjection methods for Anopheles Embryos.</a> BEI resources. , (2015).</li><li>Pham, T. B., 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=Experimental+population+modification+of+the+malaria+vector+mosquito,+Anopheles+stephensi.">Experimental population modification of the malaria vector mosquito, Anopheles stephensi.</a> PLoS Genetics. 15 (12), 1008440 (2019).</li><li>Benedict, M. Q., 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=Pragmatic+selection+of+larval+mosquito+diets+for+insectary+rearing+of+Anopheles+gambiae+and+Aedes+aegypti.">Pragmatic selection of larval mosquito diets for insectary rearing of Anopheles gambiae and Aedes aegypti.</a> PLoS One. 15 (3), 0221838 (2020).</li><li>Carballar-Lejaraz&#250;, R., James, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Population+modification+of+Anopheline+species+to+control+malaria+transmission.">Population modification of Anopheline species to control malaria transmission.</a> Pathogens and Global Health. 111 (8), 424-435 (2017).</li></ol>

With the increased availability of precise and flexible genetic engineering technologies such as CRISPR/Cas9, transgenic organisms can be developed in a more straightforward and stable way than previously possible. These tools have allowed researchers to create transgenic strains of mosquito vectors that are very close to achieving the desired properties of either refractoriness to pathogens (population modification) or heritable sterility (population suppression). However, to develop the most safe and stable genetically...

Microinjection techniques have been used to experimentally manipulate organisms since the early 1900s1. Microinjection has been used to study both basic biological functions and/or introduce important changes in the biology of a desired organism. The microinjection technique has been of particular interest to vector biologists and has been widely used to manipulate vector genomes2-11. Transgenesis experiments in arthropod vectors often aim to make vectors less efficient at transmitting pathogens by either enacting changes that decrease a vector&#39;s fitness or increase refractoriness to the pathogens they transmit. Mosquitoes transmit a variety of human pathogens and have a significant impact on morbidity and mortality worldwide. The Anopheles genus of mosquitoes transmits the human malaria parasitic pathogens,&#160;Plasmodium spp.&#160;Genetic engineering experiments with Anopheles have aimed to better understand the biology and reduce the vectorial capacity of these mosquitoes in efforts to develop novel malaria elimination strategies.
The mosquito vectors that contribute the most malaria infections worldwide are in the Anopheles gambiae species complex. However, the majority of successful transgenesis experiments have been performed on the malaria vector of the Indian subcontinent, Anopheles stephensi. While plenty of laboratory-adapted Anopheles gambiae strains exist, the number of transgenic Anopheles gambiae spp. lines reported in the literature does not compare to that of Anopheles stephensi. It is thought that the Anopheles gambiae embryo is more difficult to inject and achieve successful transgenesis than Anopheles stephensi, although the reasons for these differences are unknown. This protocol describes a method that has been proven to be consistently successful in achieving transgenesis of Anopheles gambiae embryos via microinjection. The protocol is based on a method previously developed by Herv&#233;&#160;Bossin and Mark Benedict12 with some additional details and alterations added that have been found to increase the efficiency of transgenesis.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >10x Microinjection Buffer</td>
			<td >-</td>
			<td >-</td>
			<td >1 mM NaHPO4 buffer, pH 6.8, 50 mM KCl</td>
		</tr>
		<tr >
			<td >Blotting membrane (Zeta-Probe GT Genomic Tested Blotting Membrane)</td>
			<td >Bio-Rad</td>
			<td ></td>
			<td >Neatly and straightly cut into 2x1 cm piece</td>
		</tr>
		<tr >
			<td >Conical tubes 50 ml (disposable centrifuge tube, polypropylene)</td>
			<td >Fisher Brand</td>
			<td ></td>
			<td >Ends cut</td>
		</tr>
		<tr >
			<td >De-ionized or double-distilled water (ddH20)&#160;</td>
			<td >Mili-Q</td>
			<td ></td>
			<td >In a wash bottle&#160;</td>
		</tr>
		<tr >
			<td >Dissecting microscope&#160;</td>
			<td >Leica&#160;</td>
			<td >Leica MZ12</td>
			<td >For embryo alignment</td>
		</tr>
		<tr >
			<td >Forceps&#160;</td>
			<td ></td>
			<td ></td>
			<td >No. 5 size&#160;</td>
		</tr>
		<tr >
			<td >Glass container&#160;</td>
			<td >Pyrex</td>
			<td >No. 3140</td>
			<td >125 x 65</td>
		</tr>
		<tr >
			<td >Glass slide&#160;</td>
			<td >Fisher Brand</td>
			<td >No. 12-549-3</td>
			<td >75x26 mm</td>
		</tr>
		<tr >
			<td >Incubator</td>
			<td >Barnsted Lab-line</td>
			<td >Model No. 150</td>
			<td >28 &#176;C</td>
		</tr>
		<tr >
			<td >KCl</td>
			<td ></td>
			<td ></td>
			<td >50 mM</td>
		</tr>
		<tr >
			<td >Latex dental film&#160;</td>
			<td >Crosstex International</td>
			<td >No. 19302</td>
			<td ></td>
		</tr>
		<tr >
			<td >Microinjector</td>
			<td >Sutter Instrument</td>
			<td ></td>
			<td >XenoWorks Digital Microinjector</td>
		</tr>
		<tr >
			<td >Microloader Pipette tips&#160;</td>
			<td >Eppendorf&#160;</td>
			<td ></td>
			<td >20 &#181;L microloader epT.I.P.S.</td>
		</tr>
		<tr >
			<td >Micromanipulator</td>
			<td >Sutter Instrument</td>
			<td ></td>
			<td >XenoWorks Micromanipulator</td>
		</tr>
		<tr >
			<td >Micropipette&#160;</td>
			<td >Rainin&#160;</td>
			<td ></td>
			<td >20 &#181;L</td>
		</tr>
		<tr >
			<td >Micropipette puller&#160;</td>
			<td >Sutter Instrument</td>
			<td >Sutter P-2000 micropipette puller</td>
			<td ></td>
		</tr>
		<tr >
			<td >Microscope&#160;</td>
			<td >Leica</td>
			<td >DM 1000 LED or M165 FC</td>
			<td >For microinjection</td>
		</tr>
		<tr >
			<td >Minimum fiber filter paper&#160;</td>
			<td >Fisher Brand</td>
			<td >No. 05-714-4</td>
			<td >Chromatography Paper, Thick&#160;</td>
		</tr>
		<tr >
			<td >Mosquitoes&#160;</td>
			<td >MR4, BEI Resources</td>
			<td ></td>
			<td >Anopheles gambiae, mated adult females, blood-fed 4-5 days post-eclosion</td>
		</tr>
		<tr >
			<td >NaHPO4 buffer&#160;</td>
			<td ></td>
			<td ></td>
			<td >1 mM, ph 6.8</td>
		</tr>
		<tr >
			<td >Nylon mesh</td>
			<td ></td>
			<td ></td>
			<td ></td>
		</tr>
		<tr >
			<td >Paint brush</td>
			<td >Blick</td>
			<td >No. 05831-7040</td>
			<td >Fine, size 4/0</td>
		</tr>
		<tr >
			<td >Petri dish</td>
			<td ></td>
			<td ></td>
			<td >Plastic, (60x15 mm, 90x15 mm)</td>
		</tr>
		<tr >
			<td >Sodium acetate</td>
			<td ></td>
			<td ></td>
			<td >&#160;3M</td>
		</tr>
		<tr >
			<td >Quartz glass capillaries&#160;</td>
			<td >Sutter Instrument</td>
			<td >No. QF100-70-10</td>
			<td >With filament, 1 mm OD,&#160; ID 0.7 10 cm length</td>
		</tr>
		<tr >
			<td >Water PCR grade&#160;</td>
			<td >Roche</td>
			<td >No. 03315843001</td>
			<td ></td>
		</tr>
	</tbody>
</table>

microinjection method for anopheles gambiae embryos

Embryo microinjection techniques are essential for many molecular and genetic studies of insect species. They provide a means to introduce exogenous DNA fragments encoding genes of interest as well as favorable traits into the insect germline in a stable and heritable manner. The resulting transgenic strains can be studied for phenotypic changes resulting from the expression of the integrated DNA to answer basic questions or used in practical applications. Although the technology is straightforward, it requires of the investigator patience and practice to achieve a level of skill that maximizes efficiency. Shown here is a method for microinjection of embryos of the African malaria mosquito, Anopheles gambiae. The objective is to deliver by microinjection exogenous DNA to the embryo so that it can be taken up in the developing germline (pole) cells. Expression from the injected DNA of transposases, integrases, recombinases, or other nucleases (for example CRISPR-associated proteins, Cas) can trigger events that lead to its covalent insertion into chromosomes. Transgenic An. gambiae generated from these technologies have been used for basic studies of immune system components, genes involved in blood-feeding, and elements of the olfactory system. In addition, these techniques have been used to produce An. gambiae strains with traits that may help control the transmission of malaria parasites.

A representative example of the application of the microinjection protocol described can be found in Carballar-Lejaraz&#250;&#160;et al5. The intent here was to insert an autonomous gene-drive system into the germline of a laboratory strain, G3, of An. gambiae. The system was designed to target the cardinal ortholog locus (Agcd) on the third chromosome in this species, which encodes a heme peroxidase that catalyzes the conversion of 3-hydroxykynurenine to xanthommatin, t...

Watch this Scientific Journal Video about Microinjection Method for Anopheles gambiae Embryos at JoVE.com

Microinjection Method for Anopheles gambiae Embryos

Microinjection techniques are essential to introduce exogenous genes into the genomes of mosquitoes. This protocol explains a method used by the James laboratory to microinject DNA constructs into Anopheles gambiae embryos to generate transformed mosquitoes.

Microinjection Method for Anopheles gambiae Embryos

Embryo microinjection techniques are essential for many molecular and genetic studies of insect species. They provide a means to introduce exogenous DNA ...

This protocol is significant because it provides a specific microinjection method for an Anopheles gambiae, which has been historically difficult to transform using common genetic engineering techniques. The main advantage of this technique is that it reliably creates transgenic Anopheles gambiae. The applications of this technique extend toward novel strategies for malaria elimination by facilitating the creation of mosquitoes suitable for population suppression or modification of wild-type Anopheles gambiae populations.This methodology can be adapted easily to different mosquito species. Our microinjection technology take patience and practice. There is a learning curve.To begin, use an aspirator to place 20 to 30 females in a transparent 50-milliliter conical tube. Ensure the tube is cut open at both ends and is covered with latex dental film at one end and nylon mesh and filter paper at the other, where mosquitoes will lay their eggs. Place the mosquito-filled tube in a small Petri dish filled with double-distilled water, then place the tube and dish in an incubator at 28 degrees Celsius for 45 minutes.Remove the tube from the incubator and insert the tube into an empty cage. Gently tap the tube to allow mosquitoes to fly out. Remove the tube from the cage once all the mosquitoes have flown out.When the tube is free of mosquitoes, unscrew the bottom ring, remove the nylon, and use forceps to carefully remove the filter paper with the eggs from the tube. Place the egg-laden filter paper in a plastic Petri dish containing a layer of filter paper moistened with water. Put a membrane on a clean glass slide and use clean forceps to cover the membrane with a piece of filter paper, leaving about one millimeter of the membrane filter uncovered.Wet the filter paper with deionized water, then use the brush to gently transfer 30 to 50 embryos to the edge of the membrane and align the eggs vertically along the membrane. Orient the eggs in the same direction so that when eggs are observed under the microinjection microscope, the posterior end is in a down position and forms an angle of 15 degrees with the needle. Line the entire edge of the membrane with eggs.Use a microloader tip to fill the needles with two microliters of DNA mixture. Insert the needle into the needle holder and connect the automated pressure pump tubing. Align the needle so that it makes an angle of 15 degrees with the slide's plane.Open the needle tip by carefully touching the first egg, then insert the needle's tip to about 10 micrometers in the posterior pole. A successful injection will lead to a small movement of the cytoplasm within the egg. Use the microscope coaxial stage controls to move to the next egg to continue injection.To ensure that the needle tip remains open and has not been clogged, press the Inject button before entering another embryo and visualize the small droplet at the opening of the needle. If the needle gets clogged, press the Clear button to clear the needle and repeat the droplet visualization test. Adjust the pressure as needed if the needle tip opening gets slightly bigger to ensure that the droplet size stays small.Inject about 40 to 50 eggs with one needle. After injections are complete, rinse the eggs off into a glass container lined with filter paper and filled with 50 milliliters of deionized water. Using this protocol, an autonomous gene driven-system was inserted into the germline of a laboratory strain G3 of Anopheles gambiae.The system was designed to target the cardinal ortholog, or Agcd, on the third chromosome in this species. The plasmid pCO37 is shown. It contains Streptococcus pyogenes Cas9 endonuclease under control of the regulatory elements of the nanos gene ortholog.Additionally, it has a guide RNA under the control of a U6 gene promoter and the 3XP3 CFP dominant marker cassette expressing the cyan fluorescent protein. Molecular approaches verified the accurate insertion of about 10 kilo base pairs of DNA, which was designated as AgNosCd-1. Extensive followup analyses showed that AgNosCd-1 was highly efficient at drive, created few resistant alleles, and had no major genetic load due to the integration and expression of the gene drive system.Homozygous gene drive containing mosquitoes were loss-of-function mutants, with larvae, pupae, and newly-emerged adults having a red-eye phenotype. When attempting this protocol, keeping at 88 is crucial for a good hatching to light gray eggs and avoid white eggs.

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Genetics

4.3K vistas.  University of California, Irvine. Este protocolo es significativo porque proporciona un método de microinyección específico para un Anopheles gambiae, que ha sido históricamente difícil de transformar utilizando técnicas comunes de ingeniería genética. La principal ventaja de esta técnica es que crea de forma fiable Anopheles gambiae transgénicos. Las aplicaciones de esta técnica se extienden hacia estrategias novedosas para la eliminación de la malaria al facilitar la creación de mosquitos adecuados para la supr...