Cb17 prkdcscid j mice

Donor ear or tail skin was taken from BalbC mice and transplanted onto the backs of recipient C57bl6 or NOD.CB17-Prkdc scid/J mice purchased from Jackson Laboratories (stock #000664, #001303 respectively). Mice (8 weeks, 25g) were housed in a pathogen-free facility at Arkansas Children’s Research Institute. All protocols were approved by the Institutional Animal Care and Use Committee.

Kidney organoid cultures (400,000 cells) were dissociated with Accutase, pelleted, resuspended in Advanced RPMI, and injected into the kidneys of P0 NOD.CB17-Prkdc^scid/J mice (Jackson Labs). The mice were sutured and sacrificed three weeks later and the kidneys were sectioned. To form teratomas, ∼ 2,000,000 undifferentiated hPSCs were dissociated, resuspended in 50 µl cold Matrigel (BD), and injected dorsally into 8-week old NOD.CB17-Prkdc^scid/J mice. Growths were harvested 8–10 weeks later. Male and female animals were utilized. Experiments were performed in compliance with ethical regulations and ARRIVE guidelines and in accordance with protocols approved by the Harvard Medical Area Standing Committee on Animals.

8 week old NOD.CB17-Prkdc^scid/J mice (Jackson Labs) were injected with 100μL PBS cell suspension using E1KD shScr, E1KD shILEI or E1KD shLIFR cells at 1,000, 10,000 or 100,000 cells per fat pad. Mice were evaluated weekly using calipers to assess tumor volume. Mice were sacrificed and tumors and lungs were extracted at 13–14 weeks. Final tumor volumes were weighed and lungs were stained for H&E. Briefly, lungs were formalin-fixed and paraffin-embedded in the Biorepository and Tissue Analysis Core at MUSC. Paraffin-embedded lungs were cut into 5 μm sections and stained with hematoxylin and eosin. H&E stained lungs were imaged using a 1.25x objective on an Olympus BX61 Microscope with an Olympus DP72 8-bit RGB camera and Cellsens software in the Laboratory Core in the Center for Oral Health Research at MUSC. Two-way Anova statistical analysis for volume measurements over time and Student t-test analysis of final tumor weights and metastatic tumor burden were performed using Prism 7 (Graphpad).

Animal studies were performed under guidelines established by the Duke University Institutional Animal Care and Use Committee. To augment tumor growth, 60-day continuous release 17-β-estradiol pellets (Innovative Research of America, Sarasota, FL) were implanted on the back of 10–12 week old female NOD.CB17-Prkdc^scid/J mice (Jackson Laboratories, Bar Harbor, ME). Two days later, mice were inoculated in the flank with 5 × 10⁶ BT474M1 cells in 50 % Matrigel (BD Biosciences, Bedford, MA). Biodistribution studies were initiated when tumors reached a volume of 350–500 mm³. Six groups of 5 mice were injected via the tail vein with 1.1 μCi (0.8 μg) of trastuzumab-NHS-[¹³¹I]IB-d-EEEG and 5 μCi (0.8 μg) of trastuzumab-Mal-d-GEEEK-[¹²⁵I]IB. At 4, 12, 24, 48, 96 and 144 h post injection, mice were euthanized by isofluorane overdose, dissected, and organs isolated. Blot-dried tissues of interest were weighed and counted for ¹²⁵I and ¹³¹I radioactivity along with injection standards in a dual-channel gamma counter. Results were expressed as percentage of injected dose per gram of tissue (%ID/g), except for thyroid for which %ID/organ values was determined. Tumor-to-tissue ratios also were calculated.

Female NOD.CB17-Prkdc^scid/J mice (Jackson Laboratories) were housed in pathogen-free rodent facilities at NYU Langone Health. All supplies (cages, chow, and sterile water) were autoclaved, and mice were housed in sterile conditions within high-efficiency particulate arrestance filtered micro-isolators and fed with irradiated food and acidified water. All experiments were conducted according to standard protocols outlined by the University Committee on the Use and Care of Animals. 1x10⁶ SUM149PT or olaparib-resistant clone cell lines were injected into the mammary fat pads of 6- to 8-week-old female mice. Olaparib (25 mg/kg) was administrated daily via intraperitoneal injection, and doxycycline (2 g/Kg) was administrated in the food. Tumors were monitored weekly and animals were euthanized by the end of treatment or when the tumors reached an average of 1000 mm³. Tumor volume was calculated using the following equation: volume = 0.5 × (L × W²), where L = length and W = width. All mice that reached the endpoint of the experiment were euthanized and tumors excised, weighed and proteins extracts prepared for immunoblotting.