Monitoring Endoplasmic Reticulum Calcium Homeostasis Using a Gaussia Luciferase SERCaMP

The overall goal of the following experiment is to monitor endoplasmic reticulum calcium dysregulation over time using a secreted reporter protein. This is achieved by viral vector mediated transgenesis of the target tissue, specifically liver. In this example with the Gai luciferase circa reporter next biological fluid, such as blood is collected in order to measure the amount of circa released from the tissue, then the luciferase substrate is added to the plasma samples and luminescence is measured to determine ER calcium depletion.

The results show ER calcium dysregulation as indicated by increased levels of plasma C camp ER homeostasis can be monitored over extended durations by repeated sampling of small volumes of biological fluids. The main advantage of this technique over existing methods like low affinity calcium dyes, or genetically encoded calcium indicators is that er calcium homeostasis can be monitored in vivo over time via a minimally invasive procedure. There are two therapeutic avenue for this technique.

The first is to screen for drugs that can maintain or restore er calcium homeostasis to normal physiological state. The second approach is to modify therapeutic protein to become therapeutic amps where there's secretion occurs in response to er calcium depletion. Though this method can provide insight into er calcium dysregulation in the liver.

It can also be applied to other organ systems such as brain, heart, muscle, or pancreas. After anesthetizing, a sprat dolly rat, according to the text protocol, apply ophthalmic ointment and perform a tail pinch to verify anesthetization shave the region of the abdomen slightly below the ribs to the mid abdominal region and scrub the surgical area three times, alternating Betadine and 70%ethanol. Then place the rat in a supine position on a sterile surgical area and cover with sterile surgical drapes dilute a a v gluc samp to the user optimized concentration.

In this case, we used 7.6 times 10 to the ninth viral genomes per milliliter and mixed by inverting in the tube. Then pipette 105 microliters of the diluted A a V into a sterile dish and use a 30 gauge needle to collect the virus into a syringe. Mark the target incision site to expose the liver for injection using a scalpel.

Make a horizontal incision about two to three centimeters in length below the rib cage. Blunt dissect to separate the connective tissue from the hypodermis. Cut the abdominal muscle exposing the right medial lobe of the liver.

Next, place the animal under a surgical microscope and adjust to get the right medial lobe of the liver in the field of view. Then using the syringe, inject one third the total volume of virus into three sites in the parenchyma of the right medial lobe, leaving the needle in the tissue for five to 10 seconds following injection to ensure delivery of the entire injection volume, suture the abdominal muscle and incision and use a cotton tipped applicator to apply Neosporin. Place the rat in a recovery chamber until consciousness is regained and it is able to maintain an upright position before returning the animal to a single housed home cage.

Four to seven days after injection. Prepare blood collection tubes by labeling and pre weighing them. Then at 50 microliters of 1000 units per milliliter of heparin to each tube.

After anesthetizing the rat according to the text protocol, perform a tail pinch to verify sedation and using sterile scissors. Cut the tip of the tail about one to two millimeters from the end. Collect greater than 100 microliters of blood dropwise into a tube prefilled with heparin to stop the bleeding.

Use a wet cotton applicator to apply STIC powder to the tail. Use an ethanol pad to wipe the scissors and bead sterilize in between collections. Store the blood tubes at four degrees Celsius.

If collecting subsequent samples, weigh the collection tubes and adjust the heparin to obtain a two to one ratio of blood to heparin. This step will normalize the amount of heparin in each sample. Centrifuge the tubes at 2000 times g and four degrees Celsius for five minutes.

Transfer the plasma supernatant to a fresh tube and store at four degrees Celsius for up to 72 hours or minus 80 degrees Celsius for long-term for the luciferase assay to administer thaa garin as a positive control. Dilute in ethanol to a final concentration of 2.5 milligrams per milliliter. Then inject at one milligram per kilogram intraperitoneal in the lower abdomen.

Sample the first blood sample at 24 hours after injection as described above. To measure luminescence. Prepare the selen ezine working substrate according to the text protocol and incubated at room temperature protected from light for 30 minutes.

In the meantime, thaw all samples on ice and transfer 10 microliters of plasma to an opaque walled plate using a plate reader that is capable of monitoring bioluminescence and equipped with a substrate injector. Prime the lines with substrate. Inject 100 microliters of substrate into a well.

Shake at medium speed for five seconds and measure light emission for in vitro samples. Integrate light emission over 0.5 seconds for the re step and for in vivo samples, integrate over five seconds where the read step to assess er calcium homeostasis. Using the Gluc SAR Camp Method, several controls can be included such as a constituently secreted reporter like Gluc Noag in this case.

An increase in extracellular levels of both GLUC SAR camp and Gluc noag in response to experimental conditions would be considered an ambiguous result. However, an increase in gluc SAR camp secretion with no gluc noag response supports an ER calcium dependent event. Pharmacologic modulators of the ER calcium store can be utilized to confirm the contribution of ER calcium to SAR camp release in novel paradigms.

For example, as demonstrated here dantrolene, a INE receptor antagonist that stabilizes ER calcium inhibits the release of SAR camp in response to thic argon for in vitro studies using gluc based SAR camp, the treatment induced response is best assessed over the time course. By tracking changes in relative response within a plate versus controls, it is not advisable to compare raw numbers across multiple plates where decay of substrate can lead to changes in raw values. For in vivo studies, data should be assessed independently for each animal with every animal serving as its own pretreatment control.

This is necessary to account for variability in transgene delivery and expression between animals. After watching this video, you should have a good understanding of how to utilize a sircam technology to investigate er calcium regulation in rodent models of liver disease. Don't forget that working with viral vectors can be extremely hazardous, and precautions such as wearing the proper personal protective equipment should always be taken while performing this procedure.