マイクロインジェクション ウエスタン トウモロコシ Rootworm、 Diabrotica virgifera virgifera胚の生殖細胞への変換、または CRISPR/Cas9 ゲノム編集

The overall goal of this Microinjection protocol is to modify the Western Corn Rootworm genome. This method can help answer key questions regarding Western Corn Rootworm biology and their ability to quickly overcome pest control methods such as BT Toxin and RANI. Besides Western Corn Rootworms, this method can also be applied to similar species, such as Southern and Northern Rootworms.

I first had the idea for this method when I was asked to help with injection based RANI in Western Corn Rootworm back in the 90's. Generally, individuals new to this method will struggle. Because rearing Western Corn Rootworms in the laboratory is labor intensive.

And survival rates remain low using current technology. Obtain between 500 and 1, 000 non-diapausing rootworm adults from a reliable company or research laboratory. And house them at 30 cubic centimeter cages.

Store the standard rootworm artificial diet in food storage containers about one centimeter deep. Unused diet can be stored at four degrees Celsius for up to two months. To feed the rootworm, load 100 centimeter Petri dishes with 10 to 15 grams of diet.

Load one dish of food into each rootworm cage. And replenish it when the supply runs low or the diet dries out. Also keep a 300 milliliter water reservoir, covered by a cotton ball, in each cage to serve as a water source.

Change the water when it runs low. Maintain the caged rootworm at 26 degrees Celsius with 60%humidity and a 14/10 light cycle in an insect rearing incubator. Set the lights off time later in the day such as at midnight so eggs are laid before morning work hours.

First, collect the embryos. For an egg collection surface, load a 100 millimeter Petri dish with 30 milliliters of 1%agar and water. Next, place a single layer of filter paper on the agar, followed by four layers of cheese cloth.

Each layer should be cut to size. Next, as late in the day as possible, place the egg collection plate into the rootworm cage and cover it with a tinfoil tent. Then on the following morning, remove the egg collection chamber.

Use forceps to pick up one layer of cheese cloth at a time and place into 500 milliliter beaker loaded with water. Wash the eggs off of the cheese cloth by swirling in the water. Then, use a bulb pipet to transfer the eggs to another beaker of water as a wash step.

Perform two or three washes in this manner. Then, deposit the cleaned eggs onto filter paper with as little water as possible. Next, prepare a slide by taping a piece of black filter paper flat to the slide to improve contrast.

Then drizzle fine lines of non-toxic glue onto the paper that will be used to secure the eggs. Keep the glue below the girth of an egg. While the glue is wet, use a fine brush to place the eggs onto the glue line.

Keep at least one egg's distance between each egg. Prepare multiple lines of eggs per slide. The eggs should now be injected within 30 minutes.

Now, combine the helper plasmid, donor plasmid, and phenol red buffer. Use high quality, super coiled plasmids made with an endotoxin-free commercial kit. Fortex briefly to mix.

And then spin down particulates for three minutes at maximum speed. Next, backfill pulled borosilicate glass needles with 0.5 to 1 microliters of injection solution. If any bubbles form, be sure to remove them.

Then, attach the injection needle to the micromanipulator. Now, if necessary, under the microscope, gently break off the sealed needle tip using a fine pair of forceps or by gently touching the tip into a glass barrier. Try to make the smallest possible opening that releases injection solution.

Before injecting the rootworm eggs, use a fine paintbrush to carefully wet the surface of one to three prepared eggs with sterile water. Then, inject the wetted embryos. Gently insert the needle into the middle of the embryo, and inject a small volume of solution.

Start with using 10 to 13 psi of pressure and adjust the pressure as needed. Dry embryos has a distinct round shape, by pre-wetting each embryo with water will change their shape and soften the eggshell thus allowing the needle to easily penetrate. A successfully injected embryo will have a small amount of red color.

If an egg was damaged, looks peculiar, or cannot be injected then crush it or remove it from the slide. After all eggs are injected, move the filter paper with the eggs on it to a 1%agar dish. Then, seal the dish with paraffin film and incubate it at 26 degrees Celsius until larvae hatch.

12 days after injection, start checking for larvae everyday for the next two weeks. Transfer the larvae using a fine brush to a rearing box with sprouted corn and soil to rear them under standard conditions. Germline transformation of rootworm was attempted by co-injecting a transposase carrying plasmid with a transposon carrying plasmid into pre-cellular embryos.

In the 26 survivors, each EFP expression was used to screen for donor element insertion. Although thoracent progeny were identified, screening rootworm embryos with the intense light source proved to be lethal. The next round of germline transformation utilized a red florescent protein gene driven by a constitutively active promoter from Drosophila melanogaster.

Survival rates improved, but only one DS red positive larvae was recorded. Next, embryos were micro-injected with buffer alone or with helper and thorescently marked donor DNA plasmids. A similar ratio of embryos hatched when injected with plasmids or buffer alone.

And interestingly, only about half of un-injected embryos hatched. This put the lethality of manipulating the embryos for the injection as high as performing the injection. After watching this video, you should have a good understanding of how to micro inject Western Corn Rootwall embryos.

Once mastered two to three hundred embryos can be injected per hour using this technique. This method is indebted to techniques developed for germline transformation of Drosophila melanogaster which serve as templates applicable to many other species. Following this procedure, other methods like CRISPR/Cas9 based genome editing can be performed to make targeted mutations in Western Corn Rootwall's genome.

In order to answer specific questions about genome function.