A transfecção de proteína de rato Lung

The overall goal of this procedure is to transfect protein into the mouse lung. This is accomplished by first suspending the protein of interest in project transfection reagent. Next, a mouse is anesthetized and placed on a special platform for injection.

A light source is used to aid in the insertion of the micro sprayer into the trachea. Finally, 50 microliters of fluid containing the protein of interest is injected. Ultimately, this protein transfection technique is used to show increased protein levels within the lungs of mice.

Although this method was designed to use in chronic obstructive pulmonary disease, it can also be used in other disease states such as asthma or pulmonary fibrosis. Visual demonstration of this method is critical. As the intratracheal injection steps are difficult to learn, they require a proper positioning of the mouse airway and trans illumination of the vocal cords.

Begin by preparing the protein transfection reagent first. Add 250 microliters of methanol or chloroform to the tube containing the dry project.Film. Next, vortex the tube for 10 to 20 seconds at top speed.

Once dissolved, pipette 20 microliters of project reagent into separate micro centrifuge tubes. Place these tubes under a laminar flow hood to evaporate the solvent for a minimum of six hours. At room temperature, the tubes must be completely dry.

Eye store the tubes at minus 20 degrees Celsius where they will remain good for one year. On the day of transfection, retrieve an aliquot of transfection reagent and at 50 microliters of PBS containing two micrograms of the protein of interest. Mix the solution by vortexing at low speed for three to five seconds, and then incubate at room temperature for 30 minutes before use.

When the protein is ready, anesthetize a mouse to prepare for intratracheal administration. A specially designed platform is used for intratracheal injection. It has a ramp at a 45 degree angle from its base and a metal wire hanging from two hooks at the top.

Once anesthetized, this wire is used to hang the mouse from its upper incisors. After the mouse is secured, place a light source against the trachea to aid in the visualization of the vocal cords using forceps and a paperclip. As a laryngoscope, open the oropharynx and introduce the catheter.

When using the Micros sprayer, it is important to use sufficient force when plunging on the plunger. Otherwise, instead of generating a fine mist, you'll create droplets that could potentially occlude the mouse airway. Once everything is in place, inject 50 microliters of the protein mixture through the trachea using a pen century micro sprayer.

In this example, mice were injected intratracheally with either mouse albumin or beta galacto 24 hours after protein transfection intense beta galacto. Staining designated here by the large arrows was detected within the airways of the beta galacto treated mice, but not the albumin treated mice Once mass, this technique could be performed within 10 or 15 minutes once it's performed properly. After watching this video, you should have a good understanding of how to do mouse intratracheal injection for protein transfection.