The overall goal of this method is the stable transfection of hepatocytes in vivo, with vector constructs for inducible gene or shRNA expression. This method can help to answer key questions in the field of liver research. For example, it can be applied to investigate mechanisms in liver cancer and regeneration.
The main advantage of this technique is the combined expression of CreER together with an inducible transgene R shRNA expression in mouse hepatocytes in vivo achieved by hydrodynamic tail vein injection. Demonstrating the procedure will be Eric Hubner, a med student from our laboratory. Begin by combining 150 nanograms of entry vector containing the short hairpin RNA for the targeted protein with 150 nanograms of pTC Tet vector and TE buffer to a total volume of eight microliters.
Then transfer LR clonase enzyme mix II to ice and incubate for two minutes. Vortex twice during the incubation. Then add two microliters of LR clonase enzyme mix II to the reaction and incubate at 25 degrees Celsius for one hour.
After the incubation, stop the reaction by adding one microliter of proteinase K solution. Then incubate at 37 degrees Celsius for ten minutes. Next, transform Stbl3 competent bacteria with two microliters of cloning mix using a standard heat shock protocol.
After plating the transformation on agar plates containing ampicillin, incubate at 30 degrees Celsius for 24 hours. The next day, pick single colonies and inoculate small volumes of bacterial growth medium for incubation overnight. After isolating DNA and confirming vector integrity by Sanger sequencing, prepare maxi-scale DNA preps of the construct for injection.
Begin by weighing the mice. Then prepare a sterile saline solution containing 15 micrograms per milliliter of endotoxin-free Sleeping Beauty vector construct and one microgram per milliliter endotoxin-free pchsB5. Prepare enough solution to inject each mouse with a volume that is 10%of their body weight.
Fill a sterile 3-milliliter syringe with the required volume for one mouse and attach a 27 gauge needle. After adding an appropriate amount of tissue paper to leave minimal space for movement but enough space for breathing, insert the mouse into an appropriate restrainer with breathing holes. Warm the tail using an infrared lamp for 30 to 60 seconds while watching carefully for signs of overheating such as rapid movement of the tail or other signs of agitation.
Then clean the tail with an alcohol swab. Insert the needle almost horizontally into either one of the two lateral tail veins close to the base of the tail. If placed successfully, a small amount of blood might flow back into the cone of the needle.
Inject the total volume into the tail vein within eight to ten seconds. After injection, immediately remove the mouse from the restrainer. Compress injection wound with sterile gauze for at least 30 seconds or until any bleeding subsides.
Successful hydrodynamic tail vein injection is crucial for transfection and especially dependent on injection speed, volume, and time. To make intravenous injection easier, make sure the mice are not stressed or dehydrated on the day of injection. After 30 to 60 minutes of solo housing in a recovery cage, transfer the mouse back to its original cage.
Initiate induction of Cre recombinase 10 to 15 days after tail vein injection to allow for clearance of non-integrated vectors. Dissolve ten milligrams of tamoxifen in 40 microliters of ethanol and incubate at 55 degrees Celsius for ten minutes with shaking. Vortex several times until the tamoxifen has dissolved.
Then add 960 microliters of corn oil and incubate for five minutes at 55 degrees Celsius. After cooling, draw the solution into a 1-milliliter insulin syringe with a 20 gauge needle. Then change to a 27 gauge needle.
To inject, first scruff the mouse by grabbing the neck of the mouse carefully with the thumb and the second finger, fixing the tail between the base of the hand and the fourth and fifth finger. Then intraperitoneally inject 0.1 milliliters of the solution into the left lower quadrant of the abdomen. For experiments of less than ten days, continuously administer doxycycline via drinking water using the following protocol.
First, dissolve five grams of sucrose in 100 milliliters of tap water and autoclave. Then dissolve 100 milligrams of doxycycline hyclate in five milliliters of the sucrose solution in a 15-milliliter conical tube. Next, use a 10-milliliter syringe to sterile-filter the solution through a 0.2-micron filter.
Then add the filtered doxycycline solution to the remaining 95 milliliters of sucrose solution and supply the solution to the mouse instead of drinking water. Check daily and replace immediately if the solution becomes cloudy, as this indicates bacterial overgrowth. Prepare a 1-milliliter syringe with a 27 gauge needle with one milliliter of 4%paraformaldehyde solution.
Use dissecting scissors and anatomical forceps to perform a median laparotomy and expose the liver of the euthanized mouse. Move the small intestine to the right to expose the portal vein and the inferior vena cava. Insert the needle of the prepared syringe into the inferior vena cava and cut the portal vein.
Inject one milliliter of PFA slowly to profuse the liver tissue and remove autofluorescent red blood cells. After removing the liver, rinse it in water and transfer to five to ten milliliters of 4%PFA solution to post-fix before sectioning. Injection of a transposon construct that combines the constitutive expression of an inducible Cre recombinase with inducible expression of a transgene or an shRNA of choice into Cre reporter mice showed robust expression of the GFP reporter gene after Cre recombinase activation by tamoxifen.
After doxycycline treatment, inducible transgene expression can be visualized by suitable antibodies in transfected hepatocytes. For inducible shRNA expression, cytoplasmic GFP expression detected by immunostaining can be used as a surrogate marker for shRNA expression. A transposon construct harboring a luciferase expression cassette under the control of a liver-specific promoter construct was injected.
After injection with luciferin, mice were imaged using an in vivo imaging system two weeks after hydrodynamic tail vein injection. Imaging showed robust and stable luciferase bioluminescence in the liver 15 days after injection, demonstrating that the system can be successfully used to follow the presence of transfected cells in vivo by bioluminescence imaging. Once mastered, hydrodynamic tail vein injections can be performed in about 15 minutes, while the whole procedure from cloning of our injection of mice, induction of transgene R shRNA expression to preparation of the liver will take about three to four weeks.
While attempting this procedure, it's important to keep the size and sequence integrity of the plasmids in mind. After watching this video, you should have a good understanding of how to apply the pTC Tet system with your transgene R shRNA of choice to any Crelux Pbase mouse model.
