Generation of the ZASC1 knockout mice
A ZASC1 targeting vector was cloned using a recombineering approach, described in
. Briefly, loxP sites were introduced in the intron between ZASC1 exons 3 and 4 and downstream of exon 7 with puromycin and neomycin resistance cassettes. The integrity of the ZASC1 targeting vector was confirmed by PCR amplification and DNA sequencing of cassette junctions as well as by restriction enzyme digestion. 129/J5 embryonic stem (ES) cells were electroporated (BioRad GenePulser) with the ZASC1 targeting vector and selected for integration in culture medium containing 200 μg/ml G418. G418 positive ES cell clones were analyzed for homologous recombination by Southern blot analysis at each homologous arm.
The Salk Institute Transgenic Mouse Core Facility performed blastocyst injections and highly chimeric males (by coat color) were bred with C57bl/6 females. Progeny with germline transmission of the targeted ZASC1 locus were bred with a C57bl/6 transgenic mouse expressing FLP recombinase from the human Beta-actin promoter (Jackson Laboratory Stock Number 005703). Mice with FLP recombination were then bred with CMV-Cre mice (Jackson Laboratory Stock Number 006054).
Tails collected from 14-day old pups were incubated overnight in tail lysis buffer (100 mM Tris pH 8, 5 mM EDTA, 2% SDS and 200 mM NaCl + Proteinase K) at 55°C for in house genotyping. DNA was purified by ethanol precipitation and dried pellets were resuspended in 500 μl TE. 1 μl diluted tail DNA samples were added to a 10 μl qPCR reaction mix that also included genotyping primers and FAST SYBR Green Master Mix (Applied Biosystems). Germline transmission of the ZASC1 knockout locus was detected with primer pair 5′-TCAGTGCATCAGCATACTCAAG-3′ and 5-CTGGTGGTTCTTCATCGGC-3′ and then normalized to GAPDH using primer pair 5′-ACCCAGAAGACTGTGGATGG-3′ and 5′-CACATTGGGGGTAGGAACAC-3′. qPCR analysis was performed using the Applied Biosystems ViiA 7 instrument. Genotyping for cre-recombinase, flp-recombinase and the floxed ZASC1 allele was done through a professional genotyping service (Transnetyx Inc.).
Flow cytometric analysis
Spleen and thymus samples were harvested from euthanized 4-week old animals and ground into a single cell suspension through a 70 μM cell strainer and incubated in ACK buffer (Life Technologies) to lyse red blood cells. Bone marrow samples were harvested from euthanized 10-day old animals by crushing bones and straining through a 70 μM filter. Cells were resuspended in staining buffer (3% BSA in PBS) on ice and incubated with antibodies for 30 minutes before washing away unbound antibodies. For spleen analysis, fluorescently-labeled α-CD3, α-B220, α-CD11b, α-CD4, and α-CD8 antibodies were used (eBiosciences). For thymus analysis, fluorescently-labeled α-CD4 and α-CD8 antibodies were used (eBiosciences). For bone marrow, α-c-kit, α-sca-1, α-CD105, α-CD150, and lineage markers (α-CD11b, α-Gr-1, α-Ter119, α-CD3, α-Ly6C, α-CD19, α-CD11c) were used (eBiosciences and Steve Hedrick Lab, UCSD). Samples were analyzed using the FACSAria III (BD) instrument and FlowJo software (Tree Star, Inc.).
Mo-MuLV production and titer determination
Virus was generated in extracellular supernatants from NIH 3T3-43D cells (Fan Lab, UC Irvine), filtered through a 0.45 micron filter, aliquotted and stored at −80°C. Viral titers were determined by Focal Immunofluorescence Assay (FIA) in the presence of polybrene as described previously
. Virus-infected cells were identified by staining with 538 antibody-producing supernatant (Fan lab, UC Irvine)
, and an AlexaFluor 488 Goat-anti-Mouse secondary IgG (Life Technologies). Plates were scanned and colonies counted using the Fuji FLA-5100 scanner.
The viral genome plasmid pCMMP-luciferase was used to generate the Mo-MuLV reporter viruses and mZBS-Mo-MuLV reporter virus, as described previously
. Virus-containing supernatants were harvested 48 hr post-transfection and treated with DNase (40 U/ml) before aliquotting and freezing at −80°C.
In vivo infections
Virus was harvested from extracellular supernatants from productively infected NIH3T3-43D cells and used to infect p2-p3 pups by intraperitonealinjection in a total volume of 25 μl Mo-MuLV (8×105 IU/ml). For early time points, animals were sacrificed and spleen, thymus, and bone marrow were collected in order to quantify virus infection by FIA as described above. Briefly, single cell suspensions of each tissue were generated as described before and 10-fold serial dilutions of these cells were co-cultured with NIH3T3 cells until these cells reached confluency. Similar experiments were conducted with T-cells, B-cells, and T/B-depleted samples that were purified from pooled 10-day mouse bone marrow samples using the Dynabeads FlowComp Pan-T or Pan-B cell isolation kits (Life Technologies). The cells were then stained using antibodies described in
Tumor diagnosis and histopathological analysis
For tumor studies, 2–3 day old pups were infected as described above and monitored until they developed disease. Tumors were diagnosed as described previously
. Mice were observed daily for signs of lethargy, hunched posture, and scruffy fur associated with Mo-MuLV-induced tumors. Moribund animals were sacrificed and necropsy was performed. Spleen, thymus, liver, and kidney tissues were weighed and pieces of these organs along with lymph nodes were snap frozen and fixed in either 4% paraformaldehyde in PBS (US Biological) or 10% formaldehyde.
Fixed tissues were processed by Pacific Pathology, Inc. Fixed tissue samples were embedded in paraffin and 5 μm sections were cut and studied under light microscopy after hematoxylin-eosin (H&E) staining. Additional slides were prepared for immunofluorescence analysis by rehydrating slides, performing antigen retrieval using a sodium citrate buffer, and staining with primary rabbit antibodies to CD3 (Abcam) or CD79B (Santa Cruz Biotechnology) and with a secondary anti-rabbit antibody (Life Technologies).