Ethics Statement and Animal Care
All mice were housed and handled in strict accordance with the recommendations in the “Guide for the Care and Use of Laboratory Animals” of the National Institutes of Health, Bethesda, MD. All animal experiments were carried out on National Heart Lung and Blood Institute Animal Care and Use Committee–approved protocol number H-1031R2.
Vector Construction and Production
cDNA was cloned from total RNA extracted from C57BL/6 mouse heart using the Qiagen RNEasy mini kit (Qiagen, Hilden, Germany), following first strand cDNA synthesis via the Superscript First Strand kit (Invitrogen™, Life Technologies, Carlsbad, CA). Primers mBCL2A1AFOR(1-21) 5′-ATGGCTGAGTCTGAGCTCATG-3′
and mBCL2A1AREV(519-498) 5′-TTACTTGAGGAGAAAGAGCAT-3′
were used to amplify the open reading frame of the gene and the PCR product was subcloned into the pCR4TOPO vector (Invitrogen™, Life Technologies). The human BCL2A1
gene was purchased from B-Bridge International (Clone IRATp970D1012D, Cupertino, CA). After amplification by PCR with primers introducing a Kozak sequence as well as restriction enzyme sites, the genes were subcloned into the pMIEV-IRES-GFP retroviral vector. An HA tag was also added to pMIEV-IRES-GFP vectors with the Stratagene Quick Change II site directed mutagenesis kit (Agilent Technologies, La Jolla, CA). The HA-tagged murine Bcl2a1a
and human BCL2A1
sequences were introduced into the pRRL.PPT.SF.GFPpre lentiviral vector along with the IRES sequence 
. Vectors utilized in these studies are summarized in .
Construction of BCL2A1 lentiviral vectors, and demonstration of high level expression in producer and transduced cell line.(A) Schematic of the lentiviral vectors used for expression of murine BCL2A1a or human BCL2A1.
To produce lentiviral particles, 293T cells were seeded at 5 million cells per 10-centimeter dish. Twenty-four hours later, cells were co-transfected (Calcium Phosphate Transfection Kit, Sigma-Aldrich, Saint Louis, MO) with the following plasmids: pCDNA3.HIVgag/pol.4xCTE (12 ug), pMD2.G-VSV-G (1 ug), pRSV-REV (5 ug), and 10 ug of pRRL.PPT.SF.IRES.GFPpre/HA, pRRL.PPT.SF.IRES.GFPpre/HA-mBcl2a1a, or pRRL.PPT.SF.IRES.GFPpre/HA-hBCL2A1. Vector-supernatants were collected once a day for 3 days, filtered through 0.22-um filters, and concentrated by ultracentrifugation for 2 hr at 71 900 g and 4°C in a SW28 rotor (Beckman Coulter, Atlanta GA). Pelleted viral particles were resuspended in 1 mL of StemSpan® (StemCell Technologies Inc., Vancouver, Canada) media and stored at minus 80°C. Viral titers were determined on NIH 3T3 cells via standard assays 
Cell Culture and Transduction
The murine myelomonocytic WEHI-3 (ATCC, #TIB-68, Manassas, VA) cell line was grown in IMDM supplemented with 10% FCS, 2.5 mM β2-mercapto-ethanol, glutamine, penicillin and streptomycin. Murine NIH3T3 (ATCC, #CRL-1658) and human 293T (ATCC, #CRL-11268) fibroblastic cell lines were grown in I10 media (IMDM supplemented with 10% FCS, glutamine, penicillin and streptomycin). The murine pro-B BaF3 (Leibniz-Institut DSMZ, #ACC300, Braunschweig, Germany) and murine myeloblast-like 32Dcl3 (ATCC, #CRL-11346) cell lines were grown in RPMI1640X supplemented with 10% WEHI-3 conditioned media as a source of IL-3, 10% fetal calf serum (FCS Heat-inactivated, Sigma-Aldrich), glutamine, penicillin and streptomycin (Gibco, Life Technologies, Grand Island, NY). The human erythroblastoid UT7/Epo-S1 cell line was grown in I10 media containing 2 U/mL of human erythropoietin (Amgen, Thousand Oaks, CA). Fifty thousand BaF3, 32Dcl3, or UT7/Epo-S1 cells 
were transduced with vector particles on retronectin-coated plates (Takara, Madison, WI) at a multiplicity of infection (MOI) of 5 in presence of protamine sulfate 4 ug/mL (Sigma-Aldrich).
Murine bone marrow cells were obtained by flushing bone marrow from humeri, femurs, and tibias of C57BL/6 Ly5.1 mice (C57BL/6 Ly5.1 (Pep3b) Stock 002014, Jackson Laboratories, Bar Harbor, ME). Red cells were lysed with ACK buffer (Quality Biological Inc., Gaithersburg, MD). Nucleated cells were purified using the MACS mouse lineage depletion kit (Miltenyi Biotec, Auburn, CA), and the resulting lineage negative progenitor cells were stimulated for two days at a starting density of 5×105 cells/mL in StemSpan® media supplemented with 10 ng/mL murine IL-3 (R&D Systems, Minneapolis, MN), 100 ng/mL murine IL-11 (R&D Systems), 50 ng/mL murine stem cell factor (Research Diagnostic Inc., Concord, MA), and 100 ng/mL human Flt-3 Ligand (R&D Systems). Each recipient mouse was transplanted with the equivalent of 5×105 stimulated cells that were transduced twice with lentiviral supernatants at a MOI of 5 on retronectin-coated plates in the presence of protamine sulfate 4 ug/mL.
Murine Transplantation and Follow-up
Transduced lineage negative bone marrow cells were resuspended in StemSpan® media. Following 900 rads total body irradiation each recipient C57BL/6 Ly5.2 mouse (Stock 000664, Jackson Laboratories) received transduced cells resuspended in 500 uL media via tail vein injection.
To perform secondary transplants, bone marrow cells were collected by flushing humeri, femurs, and tibias of primary mice, and red cells were lysed using ACK buffer. Using a ratio of 1 primary mouse for 3 secondary mice, primary bone marrow cells were reinfused by tail vein injection into sub-lethally irradiated secondary mice C57BL/6 (Ly5.2) (900 rads total body irradiation).
Blood was collected monthly via retro-orbital bleeding, and used for a complete blood count (Hemavet 950FS, Drew Scientific, Waterbury, CT), blood smear (hematoxylin and eosin (H&E) stained), and flow cytometric analysis.
Organs including heart, lung, kidney, spleen, liver, salivary glands, lymph nodes, any obvious tumor masses, and sternum from pre-morbid mice were collected, fixed in 10% formalin (Fischer Scientific, Thermo Fisher Scientific Inc., Pittsburgh, PA), and embedded into paraffin blocks for subsequent sectioning and H&E staining (Histosev, Gaithersburg, MD) or immunohistochemistry. Single cell bone marrow, lymph node, and spleen suspensions were obtained by flushing tissues with media. Cytospins were prepared from 105 cells, using 300 rpm for 5 minutes (CytoSpin 4 Cytocentrifuge, Thermo Scientific, Thermo Fisher Scientific Inc.). Cell suspensions were analyzed by flow cytometry. Genomic DNA was isolated from cells or tissues using Qiagen DNeasy Tissue and Blood kit according to manufacturer’s recommendation.
Flow Cytometric Analysis and Cell Sorting
Transduced BaF3, 32Dcl3, and UT7/Epo-S1 cells were sorted for GFP expression using a BD FACSAria cell sorter from BD Bioscience (San Jose, CA). Peripheral blood, bone marrow, and spleen cells were resuspended in FACS buffer (2.7 mM Potassium chloride, 1.5 mM Sodium phosphate dibasic heptahydrate, 1.5 mM Potassium phosphate monobasic, Sodium chloride 137 mM, Sodium azide 7.7 mM, 1% (w/v) BSA in water) and incubated with the following cocktail of antibodies from BD Pharmingen™ (BD Bioscience, San Diego, CA): T-cells-anti-mouse CD3e (Hamster, APC), granulocytes and NK cells-anti-mouse CD11b (Rat, PE-Cy7), B-cells-anti-mouse CD45R/B220 (Rat, APC-Cy7). Engraftment of donor cells was monitored via anti-mouse CD45.1 (Mouse, R-PE) and anti-mouse CD45.2 (Mouse, PerCP-Cy5.5). After washing with FACS buffer, cells were analyzed with a BD™ LsrII flow cytometry system from BD Bioscience. Data were analyzed using FlowJo from Treestar Inc., Ashland, OR).
Cell Cycle and Apoptosis Assays
Cell cycle status was assessed using the NuCycl™ PI Kit from Exalpha (Watertown, MA) according to the manufacturer’s recommendation. Apoptosis was assessed using the Annexin V-PE apoptosis detection kit I from (BD Pharmingen™, BD Biosciences). Analysis of cell cycle and apoptosis was performed on a BD™ LsrII flow cytometer. Data were processed using ModFit™ software from Verity Software House (Topsham, ME).
Immunohistochemical staining was performed on formalin-fixed, paraffin-embedded tissue sections from the spleen, liver, sternal bone marrow, affected lymph nodes, and tumor masses. Cut tissue sections were deparaffinized, and endogenous peroxidase was inactivated. Antibodies included rat anti-mouse B220 (clone RA3-6B2, BD Pharmingen™, BD Biosciences), rabbit polyclonal anti-mouse CD3 (Dako, Glostrup, Denmark), rat anti-mouse CD34 (Clone MEC 14.7, Abcam, Cambridge, MA), rat anti-mouse CD68 (Clone FA-11, Abcam), mouse anti-mouse Lysozyme (clone BGN/06/961, Abcam), rat anti-mouse Mac2 (Clone M3/38, Cedarlane Laboratories Limited, Burlington, NC), rabbit anti-human myeloperoxidase (MPO) with cross-reactivity to mouse (Dako), peanut agglutinin (Vector Laboratories, Burlingame, CA), and rabbit anti-human TdT with cross-reactivity to mouse (Supertechs Inc., Rockville MD). For CD3, CD34, lysozyme, Mac2 and MPO staining, antigen retrieval was performed using either the Bond Epitope Retrieval Solution 1 (ER1) or the Bond Epitope Retrieval Solution 2 (ER2) (Leica Biosystems, Newcastle Upon Tyne, UK) at 99–100°C for 20–30 min. Enzymatic retrieval was performed for CD68. TdT was kept in a pressure cooker in citrate buffer with pH
6 for 10 minutes. Subsequently, CD3, MPO, and TdT sections were incubated with the primary antibody for 30 minutes, followed by incubation with enVision + System HRP labeled polymer anti-rabbit secondary antibody (Dako) for 25 minutes. Antibodies for B220, CD34 and Mac2 were incubated with the primary antibody for 30 minutes, followed by a secondary biotinylated polyclonal goat anti-rat antibody (BD Pharmingen™, BD Biosciences) for 25 minutes and Streptavidin-HRP (Dako) for 25 minutes. Lysozyme was stained using the Animal Research Kit Peroxidase for mouse primary antibodies (Dako), as described by the manufacturer. Sections underwent colorimetric development with diaminobenzidine (DAB, Leica Biosystems), were counterstained with hematoxylin, dehydrated using graded alcohols and mounted in Cytoseal™ XYL (Richard-Allan Scientific, Kalamazoo, MI).
Frozen spleen fragments were ground in a total volume of 750 uL of RIPA buffer (Sigma-Aldrich) supplemented with a cocktail of protease inhibitors (Complete Mini EDTA-Free and PhosSTOP, Roche, Indianapolis, IN). After clarification by centrifugation for 20 min at 20 800 g and 4°C, protein concentration was determined using the Protein Assay reagent (Bio-Rad, Hercules, CA). Proteins from 14 ug of lysate were separated by electrophoresis on 4–15% acrylamide Ready gel (Biorad) in Tris/Glycine/SDS buffer (Bio-Rad). Protein standards BenchMark™ and MagicMark (Invitrogen™, Life Technologies) were used as recommended by manufacturer. Proteins were transferred to nitrocellulose membranes (Nitrocellulose Membrane Filter Paper Sandwich, Invitrogen™, Life Technologies) for 1 hr at 90 V in 10% methanol Tris/Glycine buffer (Bio-Rad). Membranes were rinsed in water, and blocked in 5% milk (Bio-Rad)/0.05% Tween20 (Bio-Rad)/PBS, pH 7.4 for 1 hr at room temperature. Primary mouse monoclonal antibodies including anti-HA (Clone 16B12, Covance, Berkeley, CA), anti-GAPDH (Clone 6C5, Ambion, Austin, TX), or anti-GFP (Clone 7.1 and 13.1, Roche) were diluted in 5% milk/0.05% Tween/PBS and incubated at 4°C overnight. Membranes were washed twice in 0.05% Tween/PBS for 10 min at room temperature. Secondary anti-mouse immunoglobulins/HRP polyclonal antibody (Dako) conjugated to HRP was diluted in 5% milk/0.05% Tween/PBS and incubated at room temperature for 45 min. After washing three times with 0.05% Tween/PBS and once with PBS, the membrane was incubated with electrochemiluminescent substrate (LumiGLOR, Cell Signaling Technology, Danvers, MA). Membranes were then exposed to a Luminescent Image Analyser (LAS-400, Fujifilm, Tokyo, Japan).
Total RNA was isolated using RNeasy mini kit according to manufacturer’s instruction (Qiagen). DNAse I (RNAse-free DNAse set, Qiagen) was added to remove any residual contaminating genomic DNA. Real-time PCR was carried out on 75 ng of total RNA per sample using the TaqMan® One-Step RT-PCR Master Mix Reagents Kit (Applied Biosystems, Foster City, CA) according to manufacturer’s instruction. Primers and probes designed to detect total murine Bcl2a1a and β2-Microglobulin were as follows: FOR-mβ2-Microglobulin 5′-CTTCAGCAAGGACTGGTCTTTC-3′; REV-mβ2-Microglobulin 5′-CGGCCATACTGGCATGCTTAAC-3′; Probe-mβ2-Microglobulin 5′FAM-TGAATTCACCCCCACTGAGACTG-3′TAMRA; FOR-mBCL2A1a 5′-GATACGGCAGAATGGAGGTT-3′; REV-mBCL2A1a 5′-GCATTTCCCAGATCTGTCCT-3′; Probe-mBCL2A1a 5′FAM-TGAACCCAAATCTGGCTGGCTG-3′TAMRA. Reactions were carried out in triplicates for each sample on an ABI PRISM 7900HT Sequence Detection System from Applied Biosystems. Copy numbers were calculated based on standard curves using plasmids harboring Bcl2a1a and β2-Microglobulin genes.
PCRs for Immunoglobulin Variable Heavy Chain Rearrangements
An adaptation of the PCR-based method reported by Schlissel and colleagues was used 
, with MuO, VH
L, and DH
R as forward primers and either J3 or J4 as reverse primers. PCRs were performed with 100 ng of genomic DNA, 16 uL of Platinum PCR SuperMix High Fidelity (Invitrogen™, Life Technologies), and 1 uL of each forward and reverse primer resuspended at 10 uM. We performed an initial denaturation step of 4 min at 94°C followed by 32 cycles of 1 min at 94°C, 1 min at 60°C, 1.75 min at 72°C, to end by a final extension of 10 min at 72°C. PCRs were loaded on 2% agarose gel in tris-acetate buffer.
Genomic DNA was digested overnight with HindIII/Sac1 (TCR) or PciI (GFP) and separated on a 0.8% agarose (Ultrapure Agarose, Invitrogen™, Life Technologies) gel and transferred overnight to a nylon membrane (Amersham Hybond™-XL, GE Healthcare, Buckinghamshire, UK). The TCRβ probe was obtained by digesting pBR322/TCRβ2 by BglII and the GFP probe was obtained by PCR performed on pRRL.PPT.SF.IRES.GFPpre/HA plasmid with SB-GFP-FOR 5′-GGCCACAAGTTCAGCGTGTC-3′ and SB-GFP-REV 5′-AGCTCGTCCATGCCGAGAGT-3′ primers. Probes were radiolabeled using a labeling Kit (Amersham Ready-To-Go DNA Labeling Beads (-dCTP), GE Healthcare) with radioactive phosphate (EasyTides Deoxycytidine 5′ triphosphate [α32-P], Perkin Elmer, Boston, MA).
Kaplan–Meier survival curves (overall survival and disease-free survival) were established using Prism 4 from GraphPad Software (La Jolla, CA). This software was also used for other statistical analyses. One-way ANOVA was used to compare more than 2 data sets. Two-way ANOVA was used to compare more than 2 data sets over time. Student t-test was used to compare 2 data sets.