This procedure visualizes and quantifies ran and firefly luciferase. In developing in vivo orthotopic tumors begin by constructing stable cell lines, expressing either ran vanilla or firefly luciferase or both. Then functionally verify the in vitro regulation of the stably expressed reporter construct.
The next step is to engraft cells onto mammary fat pad and acquire and quantify both ran and firefly luciferase readings. The main advantage of this technique over existing methods such as immunohistochemistry, is it allows for a longitudinal and dynamic view of in vivo gene regulation during tumor progression and metastasis. Start by transecting four T one memory carcinoma cells with an expression vector encoding vanilla luciferase culture.
The transfect under an optimized antibiotic concentration for several days. Then select about 10 individual ran vanilla luciferase expressing colonies to monitor the degree of expression. Continue to amplify the colonies expressing high quantities of ran vanilla luciferase for functional analyses.
Sub clone the promoter region of interest into a PGL four luciferase reporter plasmid harboring a selectable marker that is distinct from that used to select for stable vanilla expression, transfect the malignant cells and select for stable polyclonal population of firefly luciferase using traditional in vitro cell-based luciferase reporter gene assays in 96. Well plates verify that the DBR cells regulate firefly luciferase expression in a manner concordant with that observed in parental cells transiently transfected. With these vectors, place the cells on an orbital shaker for 15 minutes to lice the cells.
Also verify that CMV driven expression of vanilla is not regulated by various cell stimulations or treatment conditions. Propagate the DBR metastatic four one memory carcinoma cells with low co fluency the day before planned. In vivo inoculation passage, the cells for inoculation dissociate the four one cells by trypsin.
Wash thoroughly in growth media and dilute the cells in PBS immediately. Store on ice. Anesthetized four to six week old female bowel C mice with isof fluorine.
Using a forceps. Gently grasp and lift the fourth inguinal memory pad. Carefully place a 27.5 gauge needle bevel side up and inject into the memory fat pad directly under the nipple.
Taking special care not to push the needle into the abdominal cavity. Release the gland and slowly inject 50 microliters of the cell suspension into the memory fat pad while the mice are still anesthetized. Inject 100 microliters of red eject cine into the lateral tail vein.
Immediately place the mouse into the IVUS 200 imaging system and acquire a one minute lumin cent image. Transfer the mouse back into its cage for about an hour, which ensures that any residual ran vanilla Lucifer signal has dissipated. Now administer D Lucifer potassium salt via IP injection after five minutes, anesthetize the mouse using isof fluorine.
Transfer to the IVUS 200 in a comparable position is in the original RAN vanilla image acquisition. Using the IVUS living image software set to photons mode. Establish values for both the and firefly acquisitions.
The highly aggressive 41 cells form palpable tumors within a week. So every week inject the mice with red eject cine to monitor general tumor growth and metastasis. Then inject d Lucifer to monitor pathway specific signaling during various stages of tumor development.
Track primary tumor growth by normalizing RAN luciferase acquisitions to the first measurements at tumor cell inoculation. More importantly, calculate RLR values over time to establish the temporal regulation of individual signaling systems relative to tumor growth and progression. Now, compare pulmonary RLR values versus those calculated for the primary tumor to establish the spatial regulation of individual signaling systems relative to tumor growth and metastasis.
Dual bioluminescent images are used to visualize and track changes in the cellular composition within the primary tumor. First mixed vanilla luciferase expressing four T one cells with their non-metastatic and isogenic firefly luciferase expressing four T oh seven counterparts then inject varying ratios of these mixed breast cancer cell populations into the mammary fat pad as shown earlier. Immediately acquire both firefly and vanilla luciferase images to establish initial RS representative of a given inoculated cell mixture.
Continue to obtain longitudinal dual bioluminescent images to depict cellular composition within the primary tumor. Also evaluate the spatiotemporal metastasis of each breast cancer derivative relative to tumor growth and progression. Since each image is internally consistent and controlled, the persistent signal from the primary tumor is an important metric to gauge the overall successor failure of each vanilla and firefly data acquisition.
The linine substrate is highly sensitive to oxidation resulting in auto luminescence. Here, a failed IV injection of lanine produces non-specific signal from the intestinal tract, not the established primary tumor. In this successful renal acquisition of a mouse bearing of 41 primary tumor and pulmonary metastases pathway specific signaling measurements can be derived from imaging firefly luciferase.
These data allow for calculation of RS from both the primary tumor and its pulmonary metastases. Following the completion of this experiment, other techniques such as immunohistochemistry and in situ hybridization can be used to verify that the result in mRNA and protein are regulated in a similar fashion as your reported construct.
