The medicinal leach is a useful model system for neurobiological studies because of its simplicity, accessibility, and the possibility of static identified neurons with many different experimental tools from early embryogenesis to old age. A particularly exciting use of the embryonic lech is for time-lapse imaging studies of neuronal outgrowths in the living intact animal, following very precise modifications of gene expression in the neuron UNDERST study or its targets. In this video, we demonstrate a novel technology for fast, accurate, and highly localized istic delivery of reagents into internal layers of live embryos of the lech, using a pneumatic capillary gun that was developed in collaboration between Alex Grossman's laboratory and our laboratory.
Though this technique can be applied to the delivery of DNA double stranded RNA in any other molecule which can be loaded onto microscopic gold particles, we will use it for both the knockdown and knockin of the Axon guidance molecule.Rin. Hello, I'm from the laboratory of Eduardo Ano in the Division of Biological Sciences at the University of California San Diego. Today we are going to show you a procedure for knocking down or knocking in the expression of the Axon guidance molecule networking, which is naturally expressed in the ventral body wall, but not the dorsal domain of all body segments of the lech.
We will deliver molecules of double stranded RNA or plasmid DNA into the body wall and ganglia of selected segments at early stages of development. Holistic delivery by pneumatic capillary gun is particularly important because it enables the perturbation of gene expression patterns in selected areas of subsegment, leaving the untreated segments as internal control. Furthermore, it can be used to reach internal layers of cells at early stages of development with no damage to the tissue and without opening the specimen.
Lastly, holistic delivery has the advantage of being able to target a limited number of cells in a selected location, intermediate between single cell knockdown by microinjection and systemic knockdown through genetic approaches. So let's get started. The medicinal LE has long been used to study the properties of nerve cells and behaviorally related neuro circuits because of the simplicity of the central nervous system, about 200 bilateral pairs of neurons and a small set of giant glia makeup.
Each segmental ganglion of the 21 body segments, each neuron of the leach can be repeatedly identified and studied from specimen to specimen from the early embryo to the older adult. Since the time of the Pharaohs, the leach has been used for bloodletting purposes and its continual use in medicine makes it readily available from commercial animal suppliers. Because we study nervous system development, however, we maintain a breathing colony of leeches numbering several hundred in our laboratory, which provides us with a steady supply of embryos for our experiments.
Leeches are hermaphrodites. They have both female and male structures and cross-fertilize. Hence all specimens can lay eggs.
They do this into a structure called the cocoon, which they themselves manufacture using a set of special glands that extrude very interesting polymers in a region of the body known as the C clay tellum. A leach when ready to lay eggs, digs itself into moss, which we provide in tanks, and begins to form this egg shaped cocoon around its body when enough material has been extruded and the molecules have begun to polymerize the leach deposits eggs, as well as a nutritional yolk inside the cocoon and wiggles itself out. Using a razor blade, we can remove embryos by cutting open a window at the end of a cocoon.
Generally, each cocoon has 10 to 20 embryos in it from about the sixth day after they start to differentiate. Embryos can be removed from the cocoon and raised in pond water. It is at these stages when we usually start our experimental studies of neuronal development.
The first major step in this procedure is preparation of coated particles. In this demonstration, we will show how to deliver gold particles coated with double stranded RNA molecules of the lech homolog of the axon guidance molecule rin into the lech embryos. The target cells are ventral longitudinal muscle cells in the body wall, which are known to express and release Rin beginning at early stages in Agenesis.
Our goal is to knock down natural expression in single body segment as early as possible during development, and to observe how this affects categorizations of neurons that are sensitive to this important developmental factor. For this step, the sonication bath, vortex, benchtop centrifuge and ice bucket are needed. Also needed is 100%isopropanol, which should be put on ice before used.
Begin with 100 microliter of binding buffer that contains 50 milligrams of vault particles per milliliter. These particles are designed for RNAi and manufactured by the chell company. Next, dilute the particle solution by adding 100 microliter of binding buffer vortex, briefly and sonicate for one to two minutes to separate the particles that may have clumped together.
After the two minutes syndication, add five to 10 micrograms of double stranded RNA to the particles vortex and leave it room temperature for two minutes. Now the two minutes are up centrifuge, double stranded. RNA particles complexes at the minimum of 2, 500 RPM in the micro fuge for about 15 seconds.
To obtain the pt, remove the super natin and add 750 microliter called isopropanol with minimal disruption of the pellet. Spin briefly in micro fuge and remove the super natin after the spin versus suspend the gold particles in 100%cold isopropanol by sonicate. Briefly, two to three pulses in best sonicate to separate the particles.
Pour onto a glass slide and allow solvent to evaporate completely. Best results are obtained when particles are coated just prior to their delivery to the tissue, which is what we'll show you how to do in the next step. Before performing istic transfection, let's have a look at the experimental setup and how to prepare it.
The pneumatic capillary gun is mounted onto a micro manipulator for accurate delivery of particles into the embryos. The surface of the embryo is imaged with a video microscope. A semiconductor laser is generating a beam of light directed through the inner capillary of the gun and used for aiming at the target tissue.
Separate lines of Titan tubings connected to the three inlets of the head of the gun provide pressurized helium gas from a single source. One line is always open, creating a continuous flow of helium gas through the gun. Two other lines are normally closed with the solenoid valve and contain gold particles with two different coatings that were loaded into the tubing.
As a dry powder. The outer capillary of the gun is connected to a vacuum system with a gauge pressure of minus 12.5 PSI. The head pressure of hi is varied between 10 and 15 PSI to adjust to the desired particle penetration Depth.
Helium head pressure can greatly affect particle penetration depth, and so can the distance from the embryo to the gun. Nozzle diameter, which varies among different gene guns, is important to be chosen with respect to the target area. So now that we've shown our experimental setup, let's load our Jing gun and shoot some lech embryos.
Before loading the Titan tubing, it's critical that the coated gold particles are completely dry. The coated gold particles are first scrapped off the glass slides using a cover slip or a razor blade and ready for loading into the folded piece of whey paper. The tubing should be bent into a U shape close to the connector so that the particles will not spread along the whole tubing as they are loaded, but will be concentrated close to the connector.
Pick up the particles with a small spatula or a V-shaped piece of weighing paper and load the tubing. Flick the tubing with your finger during the loading step to spread the particles evenly. After loading the tubing, we can now move on to delivering the double stranded RNA with the Jing gun, but not before we prepare our leeches.
Here you can see our leech embryos, which are approximately 10 days old. They are now in sterile medium in which they will be kept both before and after delivery of double stranded RNA. The first step in preparing the experimental embryos for double stranded RNA delivery is adding a solution of 8%ethanol in sterile medium to a flat piece of flexible silicone with a V-shape groove.
One embryo is added and place ventral side up immediately before shooting particles. The medium in the chamber is lower just below the upper surface of the embryo, and a piece of tissue paper with a small hole cut in the middle is placed on top of it to stabilize it and to keep its surface from drying while at the same time exposing only the area that will be targeted in the embryonic stage that we are using today. Stage E 10, the longitudinal muscle cells can be located about 10 to 20 micrometers from the outer surface, internal to the epidermis, dermis, and circular muscles.
Unlike the muscle cells, central ganglia on the ventral side of the embryos in the embryonic stages, we work with E seven to E 12 are located about 50 micrometers within the ventral surface of the body. We will adjust the helium pressure so that we target the longitudinal muscles, which can be found at a more superficial depth than the ganglia. Now we are ready to shoot the longitudinal muscles with gold particles.
A load of gold particles weighing about half milligram typically can be used for up to 10 shots with a single shot usually delivering on the order of a few hundred particles. A shot is generated by briefly opening one of the valves for 0.3 seconds, causing the injection of a bolus of particles from the corresponding tubing line into the gun bore. To cover the area of tissue of interest, multiple shots are often required.
The tubing is gently tapped between shots in order to dislodge some particles and facilitate their injection into the continuous helium stream in the gun bore. If one wishes to deliver a second reagent in the same assay, you may load alternative particles to a second tubing immediately after the delivery of the gold particles. The embryos are placed back in artificial pond water, preferably one embryo per dish for one to three days until RNAi or ectopic expression is deemed sufficient.
During this time, the embryos are kept in a darkened chamber at room temperature. Here is a look at an ceto hybridization asay for narine, Mr.NA knockdown. You can see that in the treated embryo, few muscle cells do not express terrin.
These muscles contain particles. You can see that when the particles were coated with terrin double stranded RNA, they cause clearly visible knockdown of narin expression that only occurred in cells containing particles, usually one to two particles per cell. When you code particles with a plasmid encoding GFP, you get fluorescence in neuronal cells when they stopped in their nuclei with the pneumatic capillary gun we use.
In this demonstration, we were able to deliver both particles into cells in small localized volume of alive embryo without causing detectable damage to cells in the targeted area. We have also shown you how the nomadic capillary gun gives the technique of holistic delivery and new capability to knock in and knock down genes in a microscopic region of a tissue confined in three dimensions and targeted with high precision without detectable damage to the tissue. So that's it.
Thanks for watching and good luck with your experiments.
