The overall goal of this procedure is to determine gene function through the use of DNA vector-based RNA interference. This is accomplished by first designing and generating S-H-R-N-A constructs that targets specific genes. The second step is to produce lentivirus carrying the SH RNA expression cassette.
Then stable cell lines are generated by infecting cells with the lentivirus and the cells infected with the lentivirus are used in various in vitro and in vivo assays to determine the effects of silencing the gene of interest. Ultimately, depending on the assays utilized, the results can show changes in tumor genicity of the cells as a result of gene knockdown through the use of in vitro proliferation, migration, and invasion assays, as well as in vivo xenograft formation models. Demonstrating the procedure will be Daniel Stowell, ma Juan, and Daniel is a graduate student in my lab Ma Ma is a technician.
Chang is a postdoctoral fellow. This method can help answer key questions in the cancer research field, such as whether a specific gene has a role in cancer development and progression, and which aspects of these processes it may be important for. To begin design and order the oligonucleotides based on the described criteria Anil the two reverse complementary oligos that contain the 20 to 23 nucleotide target sequence in the target gene.
They will form eco R one and Hindi three sites at the two ends. After being a kneeled, another oligo contains a reverse complimentary sequence to the target sequence, and its three prime end. Neils to the three prime end of the H one or U six promoter with an upstream primer.
Use a PCR to make a fragment that has BAM H one and Hindi three sites on either end. Meanwhile digest the lentivirus vector and the PCR fragment. Then carry out a three fragment ligation reaction between the vector, the PCR fragment and the Anil oligo nucleotides.
At a one to 10 to 10 molar ratio, the resultant vector will produce the HRNA of interest with a Hindi three sequence in the loop region. If an inducible vector is used, HRNA production will hinge on the presence of an inducer molecule such as doxycycline for the Tet on system. Now transform highly efficient, competent e coli DH five alpha cells using the ligated product and identify positive vectors using a colony based PCR screen.
Amplify a product that spans the vector and the inducible promoter ligation site. Prepare plasma DNA from the positive colonies with a commercial kit. Then confirm the presence of the insert by BAM H one and ECO R one digestion and by DNA agarose electrophoresis.
Additionally, sequence the region containing the promoter and S-H-R-N-A using LIGA nucleotide P five. Using an endotoxin free MIDI or maxi prep kit, prepare the lentivirus DNA carrying the HRNA expression cassette. Determine the DNA concentration by measuring the light absorption at 260 nanometers and ensure the DN a's purity by checking that the 260 to 280 nanometer light absorption ratio is between 1.8 and 2.0.
Finally run the lentivirus plasmid on an aero gel to make sure it is S coiled Before transfection, prepare for the transfection by first slowly dropping 0.9 milliliters of solution A into 0.9 milliliters of solution B while bubbling air through solution B with a pipette. Then incubate the mixture at room temperature for 30 minutes. Next, slowly drop 0.6 milliliters of the mixture onto three different 10 centimeter plates with HEK 2 93 T cells.
Then return the cells to the incubator four to six hours later. Replace the medium with seven milliliters of complete DMEM medium. After 24 hours, add five more milliliters of medium, and after another 24 hours, harvest the medium containing the lentivirus.
Spin the harvested medium at 1500 RPM at room temperature for 10 minutes. Filter the supernatant using a 0.45 micrometer filter. Spin the flow through medium containing the lentivirus by ultra centrifugation.
Decant the supernatant to a waste container containing 5%bleach. Resuspend the lentivirus pellet in one half to one milliliter of cold one XPBS. Then divide the lentivirus solution into 50 to 100 microliter aliquots for storage at minus 80 degrees Celsius.
Finally, determine the titer of the lentivirus using a reliable technique. Typically, a 10 centimeter culture dish will produce 50 to 100 million infectious units before proceeding with cell infection. Determine the MOI of the lentiviral titer using a kit or R-T-P-C-R for this procedure.
An MOI lower than four is recommended. Begin by seeding the cells to be infected in a six well plate to 30 to 40%co fluency and culture them overnight. The next day.
Replace the medium with one milliliter of fresh, medium containing poly brain or protamine. Then add between one and 2 million iu. A lentivirus containing a fluorescent or an antibiotic marker.
Slowly shake the plate for 10 seconds to mix and incubate it for six hours. After six hours, replace the medium with normal, complete medium, and propagate the cells for two days. The media should not contain antibiotics or inducers irrespective the lentivirus used after two days.
Add the appropriate antibiotic if the lentivirus contains an antibiotic marker. Likewise, add inducer to half of the cultures if the lentivirus is inducible. After culturing the cells for two or three more days with antibiotics and inducers, carry out a western blood analysis to test the knockdown of the target gene prior to xenograft trypsin eyes.
The cells from large cell culture dishes and resuspend them in original medium containing 50%matrigel. It is essential to keep cancer cell resuspend and in 50%matrigel and the syringe on ice. Otherwise, there is a risk the matrigel will solidify and the infection will have to AB about it.
Prepare the surgical site by cleaning it thoroughly with 70%ethanol and carry out the anesthesia with excellent ventilation and an isof fluorine scavenger filter attached to the induction chamber. Anesthetize a thymic nude mice using 2%isof fluorine mixed with 1.5%oxygen three to five minutes prior to cell inoculation. Then maintain the anesthesia using 2%isof fluorine mixed with oxygen through a nose cone.
Position the mouse on a heating pad set to 30 degrees Celsius. Load the cells into syringes with 25 and a half gauge needles for subcutaneous injections, or 28 to 30 gauge needles for orthotopic injections. Five minutes after induction of anesthesia at one or two sites, inject one to 10 million cells in 200 microliter boluses.
The cell number depends on the malignancy or aggressiveness of cancer cells. For constitutively active vectors utilize two groups of mice injected with the cells expressing either the target gene S-H-R-N-A or a scrambled S-H-R-N-A fortet on vectors. Use the same two groups and divide them each into subgroups that are or are not provided inducer in their water.
Replace the docs containing water twice a week. Monitor the volume of the tumors weekly or biweekly with a C.Ensure the tumor volume does not exceed the institutional or A CUC limit and euthanize any animals that show extensive ulceration, necrosis obvious infection, uncontrolled bleeding, or end stage illness. To monitor tumor growth and metastasis via a bioluminescent marker, disinfect the work area and anesthetize the mice in an imaging chamber with gaseous anesthesia.
First, make a two to five minute exposure and check the signal intensity and then adjust accordingly. If the tumor size exceeds 1000 cubic millimeters or institutional size limit euthanize the animal upon sacrifice, document the tumor size and retain them for further analysis. This protocol was used to study the effects of why Y one knockdown on xenograft tumor formation of luciferase expressing human breast adenocarcinoma cells implanted in athymic nude mice.
The S-H-R-N-A target sequence of human YY one and a scrambled control S-H-R-N-A, which did not have significant similarity to any known human transcript were both tested. As a result, two lentiviral vectors were constructed and used to produce two lentiviruses. Both were used to infect two different cell populations and polyclonal cell populations were obtained.
After pur mycin selection. Western blood analysis confirmed the DS induced YY one knockdown in these infected cell lines. Next, the polyclonal cell population of clone three that was infected with inducible YY one S-H-R-N-A and the control.S-H-R-N-A.
LENTIVIRUSES were used to test the effects of YY one depletion on invasion. We observed that YY one depletion reduced invasiveness of the cells. Western blood analysis confirmed docs induced YY one silencing in cells with the indu YY one S-H-R-N-A, whereas cells containing control.
S-H-R-N-A did not show this effect. These cells were then used in a xenograft mouse model study compared to the control groups indu, YY one S-H-R-N-A implanted mice supplied with the dos containing water showed significantly reduced tumor formation when visualized by bioluminescence and when tumor weights were measured as expected. YY one silencing in these xenograft tumors was readily confirmed by western blot.
Studies Don't forget that working with lentivirus can be extremely hazardous according to the NIHB safety considerations for research with lentiviral vectors enhanced B safety level two containment procedures are required for all laboratory settings.
