Nod scid il2rgnull nsg mice

All mice used for 2-photon imaging and PET/CT imaging experiments were maintained at the mouse facility in MIT's Koch Institute for Integrative Cancer Research in accordance with protocols approved by the MIT Institutional Animal Care and Use Committee (IACUC). NSG (NOD/SCID/IL2Rg-null) mice were purchased from Jackson Laboratory. Alpacas (Vicugna pacos) were maintained, immunized, and bled at a farm in Massachusetts in accordance with a protocol authorized by the University of Massachusetts (Amherst) Veterinary School's IACUC and the MIT IACUC.

BL avatar. Eight- to 10-wk-old NOD-scid IL2rg null (NSG) mice (The Jackson Laboratory) were injected with BL cell lines (5–10 × 106 cells) subcutaneously into the right flank. We observed palpable tumors within 2–4 wk, depending on the growth rate of the BL cell line. Tumor volume was measured by calipers three times a week and calculated using the modified ellipsoid formula ½(Length*width2). Criteria for endpoints were tumor burden > 20% of body weight loss (BW), mean tumor volume > 4,000 mm3, and ulceration/necrosis of tumor. Tumor phenotype and EBV content were evaluated by in situ hybridization, immunohistochemistry, and flow cytometry. All animals were housed in a specific pathogen-free facility in microisolator cages and given autoclaved food and acidified autoclaved water. All animal procedures were done in accordance with the guidelines of the Institutional Animal Care and Use Committee of UMass Chan and conformed to the recommendations in the Guide for the Care and Use of Laboratory Animals (National Institutes of Health).

An in vivo medulloblastoma model was established as described before[58 (link)]. Briefly, 6–7 weeks old NOD-scid IL-2Rg null (NSG) mice (the Jackson Laboratory) were anesthetized with xylazine-ketamine, and right carotid artery was exposed. Dissociated Math1-Cre; ptch1-flox/flox cells (1×10⁶ cells in 4 μL HBSS)[58 (link)] were injected into the mouse brains using murine stereotaxic system (Stoelting Co). The coordinates were: 1.8mm to the right of bregma and 3mm deep from the dura. To disassociate the orthotopic tumor into the single cells for the serial implantation or the ex-vivo experiment, the tumor was dissected from the cerebellum, followed by digestion using Papain (10 U per mL), DNase (250 U) and L-cystein (2 mg) at 37°C for 30 min.

After the first 6 passages in SCID mice, the growth of C5 tumors was tested in alternative immunodeficient mouse strains, namely NOD-scid IL2Rg^null (NSG) mice (The Jackson Laboratory, Bar Habor, USA) and C57BL/6 pfp^−/−/rag2^−/− mice (Taconic, Hudson, USA). In addition, after passage 8, the C5 tumors of three male NSG donors were transplanted onto one age-matched male and one female NSG mouse each to determine gender differences in the tumor take rates. Furthermore, beginning at passage 6, one half of the harvested tumors was minced with the scalpel blade and ground through the cell strainer, but prepared for transient cryopreservation after washing. For this purpose, the washed pellet was resuspended with CryoSafe Medium (cryo-safe I KM-11-D, c.c.pro, Oberdorla, Germany) and stored at −80 °C. Several of these cryopreserved C5 tumor aliquots were checked for in vivo tumor growth after thawing using SCID and NSG mice.

NOD-scid IL2Rg^null (NSG) mice were purchased from The Jackson Laboratory (RRID:IMSR_JAX:005557). Mice were then bred and maintained in specific pathogen–free housing, and experiments were conducted in accordance with the guidelines of and with the approval of the Washington University Animal Studies Committee. Experiments were performed on 7- to 16-week-old male and female mice. Within each experiment, mice were age and sex matched.