マウスにおける流体力学的遺伝子送達によるタンパク質の一過性発現

The overall goal of this procedure is to transiently express genes in mice using hydrodynamic gene delivery or HGD to do this. Mice are weighed and an appropriate volume of saline containing plasmid. DNA is prepared and loaded into a syringe.

The mouse is then warmed to dilate the tail blood vessels and placed in a restrainer using the dorsal artery. As a reference, the lateral coddle veins are located and the DNA saline mix is injected into one of the coddle veins. Finally, the mouse is removed from the restrainer and placed on a warm mat for recovery.

Ultimately, results can be obtained that show transgene expressions through monitoring of blood protein levels by Western blot. The main advantage of this technique over other existing methods, such as viral transfusion expression, is that hydrodynamic gene delivery allows for the use of naked plaid DNA, which is easy to produce, safe to use, and has fewer size limitations. I'm Daniela Kovacic.

I'm a postdoc in the laboratory of Dr.Jane Raper, and I will be demonstrating this method. Begin this procedure by using a Sharpie to mark backs of white mice to indicate their experimental group reapply marks over time as necessary. On the day of the experiment, weigh each individual mouse in an animal weighing pan or bucket placed on a digital laboratory balance.

Record the weight of each mouse following the instructions in the accompanying document. Prepare enough injection mix for each mouse to be injected, plus one as proper loading of the syringes will require some extra injection mix. Use preservative and endotoxin free sterile saline that has been approved for human use.

After allowing all solutions to reach room temperature, attach a sterile 20 gauge needle to a three milliliter lower lock syringe and aspirate the DNA saline mix x. Prepare one syringe for each injection. Be sure not to generate any air bubbles.

Avoid using higher volume syringes as the fluoride of injection cannot be controlled very well and it is difficult to manipulate larger syringes in one hand. Next, switch to a sterile 27 gauge needle. Adjust the volume of the DNA saline mix as calculated.

Ejecting any excess solution back into the conical tube for later use. This will fill the needle. Without introducing air bubbles.

Do not allow the uncapped needle to touch any non-sterile surface to protect the needle prior to injections. Recap it using a one-handed technique. Place the cap on a flat surface then without holding onto the cap with the other hand, insert the needle and press the capped needle against a firm object.

To secure the cap, prepare all of the needed syringes in the same manner. Enter prior to beginning injections. Place the mouse cage with bedding included on a heat pad for five minutes so that the temperature of the bedding is approximately 38 to 39 degrees Celsius.

This will dilate mice blood vessels. Note that anesthesia is not recommended for this procedure while the mice are warming, use laboratory tape to immobilize a dorsal access restrainer on a flat surface. After five minutes have passed, uncapped the syringe to be used for the first mouse injection.

Place the syringe on the cap to prevent the uncapped needle from touching unclean surfaces. For both safety and cleanliness. Keep one needle uncapped at a time.

Next, pick up a mouse by the tail. Place it gently into the restrainer and insert the plug. Use as little restraint as necessary to keep the tail immobilized.

Ensure that the mouse is breathing freely. Next, locate the lateral coddle veins by looking straight down at the back of the mouse. The red line running in the middle of the tail is an artery.

While the blue lines on either side of the tail are the lateral coddle veins, position the mouse, so one of the lateral coddle veins is visible. Then wipe the tail of the mouse thoroughly with an alcohol swab. Using the non injecting hand hold the tail tightly between the index and middle fingers so that the tail passes under the index finger over the middle and third fingers and under the little finger.

Allow the thumb to remain free to move. Using the other hand, use an alcohol swab to wipe the injection area. Again, allow the area to dry.

Now with the injecting hand, pick up the loaded syringe and hold it by the barrel between the thumb and the other four digits. Do not hold onto the plunger. Position the syringe parallel to the tail with the needle pointing toward the body of the mouse and the bevel or slanting edge of the needle facing up.

Insert the needle into the tail vein. If the vein has been correctly located, the needle will slide in with no resistance while holding the needle steady, use the thumb of the tail holding hand to close down on the hub of the needle and press the hub against the tail to hold the needle in position. Do not apply extreme pressure and do not hold onto the needle shaft, as these will impede the flow of liquid into the vein.

Next, reposition the syringe holding hand so that the index finger and middle finger are holding onto the flange and the thumb is on the end of the plunger. Press down on the plunger in one continuous motion and inject the full volume of liquid in six to eight seconds. Remove the needle from the vein and with a tissue, apply gentle pressure to the tail to stop any bleeding.

Here the injection process is shown in real time. Note that full insertion of the needle is not recommended due to an increased risk of shearing of the vein Following the injection, remove the mouse from the restrainer and place it in a recovery cage placed on top of a heating pad. If the injection room is cold, this step is critical for mouse survival.

Hold onto the tail of the mouse until bleeding stops completely. Observe the mouse for one hour following hydrodynamic. DNA delivery.

An initial period of panting and immobility is normal, but make sure that the mouse shows signs of recovery. In approximately five minutes after the mouse has recovered, return it to the housing cage and ensure that it has an abundant supply of food and water. Next, we'll demonstrate tail injection using an alternative hand position.

This method is particularly useful when it is necessary to inject mice closer to the base of the tail. For example, when correcting a failed injection on the same day or when injecting mice with shorter tails, Prepare the mouse for injection and position it in a restrainer. As before, rest the non injecting hand on a flat surface with four fingers bent at a 90 degree angle, the fingers should rest on top of each other.

Creating a platform, hold the tail of the mouse between the thumb and the pointing finger. Using the other hand, wipe the area to be injected with an alcohol swab. Allow the area to dry.

Grab the injection syringe with the I injecting hand and hold it by the flange and the end of the plunger ready for injection. Then position the syringe parallel to the tail with the needle pointing toward the body of the mouse and the bevel of the needle facing up. Inject as before to assess whether the addition of a hiss tag at specific sequence locations interferes with correct processing and secretion of the baboon APO.

L one protein Mice were injected with a plasmid carrying one of three differentially hist tagged baboon APO L one genes, and another plasmid carrying baboon HPR. The success of the injections was evaluated by Western blood analysis of circulating HPR levels in mouse blood two days post-injection. As shown here, baboon HPR was highly expressed in the plasma of all, but one of the transfected mice indicating that the HGD was successful.

Minor drops in expression levels as seen in one of the two mice in B APO L one, his tag group one are generally attributable to a very small one to two second reduction in injection speed. Here, plasma sample lane one shows a complete lack of protein expression. This indicates a failure of HGD most likely due to incomplete injection of the full volume of the delivery vehicle to assess the effect of his tagging on B APO L one secretion expression of the hiss tag was also evaluated as seen here.

There was a complete lack of detectable hist tagged protein in groups one and two. The only group of mice that had detectable levels of the protein in their plasma was group three. Taken together this data indicates that the lack of his tagged protein in groups one and two is the result of differences in gene expression or protein processing rather than a failure of the injections.

It also highlights the importance of including a tracer protein in the injection mix To monitor the success of the HGD. After watching this video, you should have a good understanding of how to prepare the mice and the syringes and how to insert the needle into the tail vein for rapid injection of temid DNA to express genes and mice by hydrodynamic gene delivery. The most difficult aspect of this procedure is maintaining the needle in position throughout the injection.

The correctly inserted needle may sleep out of position due to accidental movement by either the injecting person or the non anesthetized mouse to increase the chance of success. We avoid injecting mice that are over 27 grams in weight as handling of a syringe that has been loaded to three ml. Capacity is more difficult.

We find that mice weighing 23 to 27 grams are the most convenient to inject. The veins of mice weighing less than 20 grams may be too small to inject.