Name: Microinjection of A. aegypti Embryos to Obtain Transgenic Mosquitoes (Video) | JoVE Journal
Uploaded: 2007-07-04T00:00:00.000+00:00
Duration: 17 min 39 s
Description: Learn microinjection techniques for generating transgenic Aedes aegypti mosquitoes. This method demonstrates needle preparation, embryo desiccation, and genetic modification to advance vector biology research.

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5 minutes

Preparation in advance of microinjection
<ol>
<li>Blood feed mosquitoes: for injection from Monday to Friday feed females on the previous Thursday.</li>
<li>Prepare glass needles using program 3 or 2 (see Materials).</li>
<li>Laying tube for the embryos is Drosophila culture vial. Wet cotton wool in the bottom, with a wet disk of filter paper covering it.</li>
<li>Prepare plastic cover slip by sticking double-side tape to one end. Trim tape to cover slip so that it ends at edge of cover slip.</li>
<li>Prepare oil for desiccation (see Materials)</li>
<li>150 ml plastic beaker with a wet cotton wool in the bottom, covered with a wet disk of filter paper (80mm Whatman) to keep embryos after injection.</li>
</ol>
Set up of forced laying
<ol>
<li>Collect 6-10 blood fed females with the use of an aspirator and transfer them to the Drosophila culture vial with wet cotton and filter paper.</li>
<li>Put the mosquitoes back into insectory conditions in the dark, and allow to lay eggs for 1h and 15min.</li>
<li>Let adults fly into the cage and remove the filter paper disk with embryos.</li>
<li>To line up eggs, do so under dissecting scope. Using fine forceps No. 5 or a fine paintbrush, pick gray to darkish-grey embryos and arrange in line on square of 3MM Whatman paper soaked with water. All embryos must be in the same orientation, as injection has to be at the posterior pole. The anterior end of the embryo is slightly wider than the posterior. Dry the filter paper with embryos by pressing dry filter paper to it (wet things won't stick to tape). To transfer the eggs, invert the slide containing the double-sided tape and gently press against the eggs. The posterior end of the eggs have to be very close to the edge of the double sided tape.</li>
<li>Desiccate the embryos about 1 min. at room temperature. They start to dimple slightly as they dry.</li>
<li>Cover desiccated embryos with halocarbon oil to prevent further desiccation.</li>
</ol>
Microinjection of the embryos
<ol>
<li>The most important aspect of good injection is the quality of the needle (see Materials).</li>
<li>Fill a needle with the DNA solution to be injected by using a microloader. Very little injection solution is needed: 1 to 2 &mu;l.</li>
<li>Connect the needle to the Transjector, which controls the injection time (Manu) and pressure (600), and the backpressure (250). Microinjection is performed using a microscope with a moving stage (Leica) at x 10 magnification and micromanipulator. If necessary, a raised microscope stage may be prepare by stacking 4-5 microscope slides. Place the cover slip carrying the embryos onto raised stage. Inject embryos horizontally to prevent tearing of the endochorion; penetration should be at the posterior pole. For injection, I keep the needle stationary and move the stage of the microscope.</li>
<li>Inject 0.2-0.5 nl of solution, corresponding to approximately 1-5% of the embryo volume. Control the injection volume by adjusting the injection pressure and time. The slightly bowed, desiccated embryos should regain their turgid appearance following injection.</li>
<li>Pick embryos from the cover slip using fine forceps or a fine brush, and place them in a plastic beaker with wet cotton and wet filter paper. Cover beaker with tissue paper. Keep in place with elastic band around the rim and return to insectary conditions for 4 days.</li>
</ol>
Post-injection
<ol>
<li>Four days after, carefully place embryo paper, embryo-side down, on the water in large plastic box with a small amount of food. Embryos hatch over a very long period: don't throw out the culture until at least 4-5 weeks post-injection.</li>
</ol>
Notes
DNA for injection
Plasmid DNA solution for injection was isolated using EndoFree plasmid kit. Construct and Helper plasmid mix together in right concentration, precipitated with izopropanol. The pellet was washed with 70% ethanol before resuspending in injection buffer. Before injection, clean DNA using Millex-GV column.

<table><tbody><tr><td>1x Injection solution</td><td>Buffer</td><td></td><td></td><td>5mM KCl, 0.1mM sodium phosphate, ph 6. 8</td></tr><tr><td>Construct plasmid</td><td>DNA</td><td></td><td></td><td>0.5 mg/ml. </td></tr><tr><td>Helper plasmid</td><td>DNA</td><td></td><td></td><td>0.3 mg/ml </td></tr><tr><td>EndoFree Plasmid kit</td><td>Kit</td><td>Qiagen</td><td></td><td></td></tr><tr><td>Millex-GV column</td><td></td><td></td><td>SLGV RO4 NL</td><td>To clean DNA.</td></tr><tr><td>Glass puller</td><td>Instrument</td><td>Sutter Instrument Co.</td><td>Model P-87</td><td>Heat-815, Pull-10, Vel-10, Time-100.Heat-700, Pull-30, Vel-36, Time-50</td></tr><tr><td>Quartz puller</td><td>Instrument</td><td>Sutter Instrument Co.</td><td>Model P-2000</td><td>Heat-275, Fil-3, Vel-38, Del-250, Pull-141</td></tr><tr><td>Injection glass needles</td><td></td><td></td><td></td><td>Prepared by drawing capillary tubing into a fine 3-8 um with a shaft of approximately 100-300 um micropipette puller. When the needle is new, the tip is sealed and usually breaks in the first or second injection or the glass needle tip can be beveled using a Brown type micro-pipette beveller (Sutter Instrument).</td></tr><tr><td>Aedes aegypti</td><td>Animal</td><td></td><td></td><td>Male and female mosquitos, to obtain embryos.</td></tr><tr><td>Drosophila culture vial</td><td></td><td></td><td></td><td>For mosquito egg-laying.</td></tr><tr><td>Halocarbon oil</td><td>Reagent</td><td></td><td></td><td>Oil for desiccation: Halocarbon oil 700:Halocarbon oil 27(1:1)</td></tr><tr><td>Forceps No5</td><td>Tool</td><td></td><td></td><td></td></tr><tr><td>Fine paintbrush</td><td></td><td>Kolinsky Sable</td><td>No. 0000</td><td></td></tr><tr><td>Whatman paper</td><td>Tool</td><td></td><td></td><td></td></tr><tr><td>Microloader</td><td>Tool</td><td>Eppendorf</td><td>5242 956.003</td><td></td></tr><tr><td>Transjector</td><td>Tool</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>Microscope</td><td></td><td>Leica Microsystems</td><td></td><td>With moving stage and micromanipulator.</td></tr><tr><td>3MM Whatman paper</td><td></td><td></td><td></td><td></td></tr><tr><td>double sided tape</td><td></td><td></td><td></td><td></td></tr></tbody></table>

microinjection of a. aegypti embryos to obtain transgenic mosquitoes

Microinjection of A. aegypti Embryos to Obtain Transgenic Mosquitoes

In this video, Nijole Jasinskiene demonstrates the methodology employed to generate transgenic Aedes aegypti mosquitoes, which are vectors for dengue fever.  The techniques for correctly preparing microinjection needles, dessicating embryos, and performing microinjection are demonstrated.

In this video, Nijole Jasinskiene demonstrates the methodology employed to generate transgenic Aedes aegypti mosquitoes, which are vectors for dengue fever. The techniques for correctly preparing microinjection needles, dessicating embryos, and performing microinjection are demonstrated.

In this video, Nijole Jasinskiene demonstrates the methodology employed to generate transgenic Aedes aegypti mosquitoes, which are vectors for dengue ...

I am ya. I am working in Anton James'lab. Today I'm going to show you how to do microinjection into ADIs Egypt embryos for a purpose of getting Transgenic mosquitoes.To get the Embryos for micro injections, we need to have female fed on the blood. Females supposed to be fed on Thursday. If you want to do a microinjection from Monday to Friday, this is also a vile.I'm taking the cotton and put in and pour the water. Cotton's supposed to be wet and take three mm whiteman circle, put on the top and push down. And I need to pour the water, clean roof, napkin, and close with cotton.This is ready Right now I am going to collect six 10 females and I transfer females to to withdraw for little while and I'm going to put them into dark conditions and put in the chamber, which has a 27 degrees celam for one hour and 15 minutes. I am making needles for Micro injection using glass needles and we using polar shuttle instrument corporation and model P 87. I place a glass needle into the ring and fix from both sides.Close the lid and push pull needle is red. Now I'm going to show you how to do be link of the needles. The needles after pulling are closed and we need to reopen the needles.There are two ways to do it. One way is be link and another way just touch the embryo before injections. Okay, this is Lin way.I have the needle and fix this needle here. Here is un above the tip we cannot use for micro injection. This tip for micro injection, we need to have open tip.This is the wheel. I try to touch the surface of the wheel with the needle and the needle tips have to touch the surface of the wheel. When the tip touch the surface, you have to put wheel on.Here you can see the needle is ready for microinjection. The the needle tip is be and you can see that the tip is shining. This, this means the needle is open and ready for microinjection.Remember, just the end of the tube have to be broken. If you make a big hole, the needle is not good. The hole Have to be very small.One hour and 15 minutes passed And embryos should be ready for micro injections I'm taking from the chamber. Take you a while and you can see embryos, a black dose on the paper and right now I'm transferring females to the cage. Just let them fly in.Now I'm going to show you the tools that I'm going to use for alignment embryos. This forces I'm using to take X paper from wild. This forces I'm using to do line of embryo.The Caesar, we need to to cut double stick tape and this forces I'm using to take out eggs after micro injections. This is the cover slip. To this cover slip.I attach a double stick tape to pick up embryos from the watman paper. Now I'm going to align embryos. The first I'm taking X paper from the while.You have to remember that the paper all time have to be wet. I am going to line embryos from gray to dark gray. This is good stage for injections.Don't take white embryos and black, all embryos have to be in the same orientation. We have two poles, Ontario and poster we're going to inject in Posterial.End. Ontario side is a little bit wide than posterior.This is cover slip. I'm going to make A cover slip ready to pick up embryos. For this, I need double stick tape attached to the cover.Slip, cut, ages and cover slip is ready. Right now I need to to dry X paper very well because if embryos are wet, they never stick to the tape. For this, I am using dry free MV.Right now I'm taking cover slip and double steak tape and press to the the embryos very slightly.And I have embryos on the cover slip. And right now is this dication step. This dication step will mostly have to wait in this position about one minute maybe.But you have to look by microscope and you can see the eggs start to shrink. These embryos are desiccated. You can see the dimples and when you see these dimples you have to put oil immediately and stop desiccation.When embryos are desiccated you have to put oil immediately. Embryo are ready for microinjection And I'm putting line embryos on the stage and I'm going to fill needles, DNA for this. I'm using append pipe and loading needle also from append from here.And I'm taking my DNA taking 1.5 microliter. Okay and load and the needle and I fixed needle to the holder. And hol is connected to the micro injector.We are using einor micro injector, which call Femto jet. Now I'm ready to inject the embryos. For this papers we are using injection pressure 600 back pressure 250 and time is manual.Right now I see very perfect embryos in the under microscope. And after that I move needle in and also I have to see very perfectly and focus needle in the microscope. After that, I put back embryos and can see embryos and needle in the same field.And right now attach needle very close to the posterior end of the embryos. And I am doing injection to a posterior end of the embryos. And first what I'm doing, first I have to see very clear embryos in the microscope.I move the stage of microscope and always see where the needle not moving. Good injection. The perfect injection.When you see the posterior end and you reach a needle, touch the posterior and you penetrate it and you cannot see any shrunk of the X, any shrunk like that. If you do like that, it's not very good. But if x still in the position like this and you penetrate the needle, It Means your needle is very good and injection is very good.After microinjection, I need to transfer the EMREs in, in the wet conditions. For this, I'm using plastic chamber with wet cotton on the bottom and wet paper. I put this my on the cotton and be sure that this paper is wet.This is ready. I'm taking my forcet. I am teaching the embryos under oil and putting on the wall on the paper.Please don't put on the bottom because oil have to go down from the embryo and I, I'm taking 2, 3, 5 embryos in in one time or one it's also okay. Now I'm going to prepare embryos to be ready for insectary. For this I am taking kin wipe, cover the embryos and fix with rubber.And this is Ready for insect for five days. Next we keep in the Inspect for five days and after that we're going to hatch with embryo, eh, and get the transgenics. Now I'm in ECT and I'm going to show you how to get hatch the embryos.And the first I'm going to pour water in the plastic chamber and add the food. Couple drops, take the embryos, which stay in a insectory food five days. Take the egg paper, do small hole and put embryos down.That's it. Embryos start hatch after first in the first week. And also some embryo start hatch after four weeks.And we have always in the sandage, different stages of larva and and pu, I just have showing for you how to do microinjection into a dete embryos. And I want to say three steps is very important in this procedure. You have to make very good needles.You have to pick up embryos in the right stage. Dessication have to be done also very precisely, not too much. In about four weeks we'll have transgenic mosquitoes.

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