Nod cg prkdc scid il2rg tm1wjl szj nsg female mice

Five x10⁶ cell/ml of parental, hCEACAM1 and hCEA-expressing MDA-MB-231 cell lines were harvested, washed twice with PBS and resuspended 1:1 in PBS matrigel (Corning). 500 000 cells from each cell line were injected into the mammary fat pad of eight week-old NOD.Cg-Prkdc scid Il2rg tm1Wjl/SzJ (NSG) female mice (Jackson Laboratory) (n = 3), previously anesthetized with isoflurane.

Unless otherwise described, mice were generated on a C57BL/6 background. Mice with a conditional Erg knockout allele (Erg^fl) were generated as previously described (23 (link)). Mice carrying a conditional c-Myc knockout allele (24 (link)) (Myc^fl) were obtained from the Jackson Laboratory (Myc^tm2Fwa). These mice were interbred with either Rag1Cre mice (28 (link)), in which Cre recombinase is expressed during lymphopoiesis from the CLP stage (57 (link)) or CreERT2 mice (29 (link)), in which the expression of the Cre recombinase can be initiated by TAM treatment to generate Rag1Cre^T/+;Erg^fl/fl, Rag1Cre^T/+;Myc^fl/fl, CreERT2^T/+;Erg^fl/fl, CreERT2^T/+;Myc^fl/fl, and wild-type control littermates. Subsequently these were crossed to P190 transgenic mice (13 (link)). Mice were co-housed in a barrier facility and analyzed from 6 to 18 weeks of age. Male and female mice were used. The primers used for genotyping are provided in table S4. NOD.Cg-Prkdc^scidIL2rg^tm1Wjl/Szj (NSG) female mice were obtained from the Jackson Laboratory and co-housed in individually ventilated cages in a specific pathogen–free facility. Experimental procedures were approved by The Walter and Eliza Hall Institute of Medical Research Animal Ethics Committee.

Tumor tissue and malignant PE cells were obtained from the patient at nephrectomy and thoracentesis, respectively, using the UC Davis Internal Review Board approved protocol. Tissues and cells were transferred upon collection to The Jackson Laboratory (Sacramento, CA) where all animal experiments were performed, in accordance with the Animal Care and Use Committee of the Jackson Laboratory and conformed to the recommendations in the National Academy of Sciences Guide for the Care and Use of Laboratory Animals (9 ). The tissue was dissected into small fragments (approximately 3 mm³) and implanted subcutaneously to the flank of 10 healthy 6 to 8-week-old NOD.Cg-Prkdc^scid Il2rg^tm1Wjl/SzJ (NSG) female mice (The Jackson Laboratory) using a 10-gauge trocar. Tumor cells were isolated from the PE by centrifugation, then inoculated subcutaneously (1.0 × 10⁷ cells per injection) to the flank of 10 additional NSG mice (PE-P0). Once the PE-P0 tumor size reached 1000 mm³, the tumors were harvested, fragmented into 3 to 4 mm³ sections, and transplanted into recipient mice (PE-P1) for drug efficacy studies. Tissue from the patient primary tumor, patient PE, renal tumor xenograft, and PE-P0 xenograft were submitted to the UC Davis Department of Pathology and Laboratory Medicine for standard histologic processing, embedding, and H&E staining.