A variety of transgene delivery methods (e.g., viral, liposomal, or stem cell delivery) can be imagined for the analysis of a DD fusion protein in an animal model. In this protocol, we discuss using tumor xenografts in mice as a mechanism for delivery of our destabilized protein.
Before using an animal model system, ligand-dependent stability of the fusion protein of interest should be validated first in heterogeneous or clonal cell populations in tissue culture (see the related CSH protocol – Hagan, E. L., et al). Always confirm that the protein of interest can retain its function either an N- or C-terminal fusion. Briefly, create a stable cell line carrying the DD fused to a protein of interest (POI). This cell line should be tested for Shield-1 dependent protein levels by performing a dose-response experiment using varying concentrations of Shield-1 (from 3 μM to 1 nM) and a time course assay. Typical in vitro assays for protein levels such as immunoblotting or ELISA and a functional assessment of the DD fusion protein should be used. Maximum stabilization typically has been observed using 1 μM Shield-1, with maximum protein levels achieved after anywhere from 4 to 24 hours, depending on the protein of interest. Upon removal of Shield-1, protein is degraded to background levels within 2–4 hours. Shield-1 stabilization results in over a 50-fold increase in mean fluorescence intensity of yellow fluorescent protein (Banaszynski et al.) and a 6-fold increase in luminescence of a thermo-stable luciferase (tsLuc, Tisi et al.) in vitro.
Shield-1 stabilization displays similar dynamic ranges in living mice, however it is reasonable to expect that a large increase in dosage is necessary to achieve sufficient levels of Shield-1 at the xenograft site. For animal experiments, a range of Shield-1 dosages (1–10 mg kg−1) should be tested for optimal stabilization of destabilized transgenes. Maximum stabilization of DD-tsLuc is typically seen 8–24 hours after Shield-1 injection. Destabilized proteins return to basal levels by 36–48 hours after injection. It is advisable to run a pilot experiment with fewer groups and low numbers (2–4) of animals per group to determine a suitable Shield-1 dosage regimen and to optimize transgene detection assays.
Day 1: Plate Cells
(1) Plate cells containing a stably integrated destabilized transgene in a culture flask (175 mm3). Grow the cells to 80% confluence and count number of cells. Adjust the number of cells to implant per animal depending on the growth of the cell line in animals. Typically 100,000-5 million cells per animal should be sufficient for detection of the DD-POI after a few days. Normal experimental group sizes (n = 6–8 mice) should allow for statistically significant comparisons.
Day 2: Transplant Cells
(2) Trypsinize, quench with complete media, and spin cells (5 min, 500 g, swing-bucket rotor). Wash cells three times with PBS, spinning as above. Resuspend cells in 100 μL (10,000 cells per μL) of DMEM (no FBS) per animal.
(3) Xenograft cells subcutaneously (or at desired location) into mice anesthetized with isoflurane (2%).
Day 5: Shield-1 Treatment
(4) Wait several days to allow cells to form stable grafts and begin experimental protocol. Reconstitute Shield-1 in DMA at various concentrations up to 10 mg mL−1. This stock solution may be kept for several months at −20 °C. Make up a fresh solution of 9:1 PEG400:Tween 80 before each injection. Inject Shield-1 i.p. at a concentration of 10 mg kg−1 or 3 mg kg−1 using a 1:1 mixture of 10 mg mL−1 or 3 mg mL−1 Shield -1 in DMA stock solution with the fresh 9:1 PEG: Tween mix. Inject control mice with DMA/PEG/Tween vehicle alone. Shield-1 may be injected intravenously, however intraperitoneal injections often produce more reliable results.
Day 6 and later: Experimental Analysis
(5) Assay for experimental DD-POI stabilization. For direct protein measurement, remove tumors, standardize tumor tissue amount (typically 1 g per mouse), homogenize tissue and assay for protein levels via ELISA or immunoblotting.
(6) Continue dosing with Shield-1. To maintain high levels of DD-POI, dose every 48 hours.
(7) Periodically assay directly for DD-POI stabilization and for the phenotypic or functional effects of protein stabilization. For instance, we assayed for tumor xenograft regression based on Shield-1 stabilization of the secreted IL-2 protein. We monitored subcutaneous tumor size via caliper measurements.
Interpretation of Results
(8) A negative control group receiving a transplant of xenografted cells that do not contain DD-POI but are dosed with Shield-1 will help attribute observed results to stabilization of the transgene, and not any nonspecific effects of the ligand, vehicle, or xenograft procedure. A negative control group of mice receiving xenografted cells containing DD-POI that are given vehicle alone will show the background level of destabilized protein activity and provides a comparison for groups in which the DD-POI is stabilized by Shield-1. A positive control group in which mice receive cells containing unregulated POI will allow observation of transgene effects without temporal and tunable ligand control. Different doses of Shield-1 (10 mg kg−1 and 3 mg kg−1) can be used to determine any concentration-dependent activities of the protein of interest. Also, different dosages can affect the systemic diffusion of a secreted transgene. For example, 10 mg kg−1 Shield -1 can stabilize secreted IL-2 such that it can be detected systemically, while at a dose of 5 mg kg−1, IL-2 is only locally detectable at the xenograft site.