- Methodology article
- Open Access
A tamoxifen inducible knock-in allele for investigation of E2A function
BMC Developmental Biologyvolume 9, Article number: 51 (2009)
E-proteins are transcription factors important for the development of a variety of cell types, including neural, muscle and lymphocytes of the immune system. E2A, the best characterized E-protein family member in mammals, has been shown to have stage specific roles in cell differentiation, lineage commitment, proliferation, and survival. However, due to the complexity of E2A function, it is often difficult to separate these roles using conventional genetic approaches. Here, we have developed a new genetic model for reversible control of E2A protein activity at physiological levels. This system was created by inserting a tamoxifen-responsive region of the estrogen receptor (ER) at the carboxyl end of the tcfe2a gene to generate E2AER fusion proteins. We have characterized and analyzed the efficiency and kinetics of this inducible E2AER system in the context of B cell development.
B cell development has been shown previously to be blocked at an early stage in E2A deficient animals. Our E2AER/ER mice demonstrated this predicted block in B cell development, and E2AER DNA binding activity was not detected in the absence of ligand. In vitro studies verified rapid induction of E2AER DNA binding activity upon tamoxifen treatment. While tamoxifen treatment of E2AER/ER mice showed inefficient rescue of B cell development in live animals, direct exposure of bone marrow cells to tamoxifen in an ex vivo culture was sufficient to rescue and support early B cell development from the pre-proB cell stage.
The E2AER system provides inducible and reversible regulation of E2A function at the protein level. Many previous studies have utilized over-expression systems to induce E2A function, which are complicated by the toxicity often resulting from high levels of E2A. The E2AER model instead restores E2A activity at an endogenous level and in addition, allows for tight regulation of the timing of induction. These features make our E2AER ex vivo culture system attractive to study both immediate and gradual downstream E2A-mediated events.
E2A, encoded by the tcfe2a gene and a member of the E-protein family, is a basic helix-loop-helix (HLH) transcription factor critical for regulating gene expression in several developmental systems . Originally identified as an immunoglobulin (Ig) enhancer binding factor, E2A has since largely been studied for its roles during the development of B and T lymphocytes [2, 3]. E2A participates in various aspects of lymphocyte development including lineage commitment, initiation of lineage specific gene expression, rearrangement of B and T cell receptor genes, and differentiation through multiple developmental stages. In addition to roles in lineage commitment and cell differentiation, E2A has also been shown to regulate cell cycle, proliferation, and survival.
E-proteins, including E2A, HEB, and E2-2 in mammals, function as dimers to bind DNA and regulate gene expression. E2A homodimers are the primary E-protein dimers functioning in B cells, whereas E2A-HEB heterodimers are the primary dimers functioning in T cells [4–6]. Recent data suggests that E2-2 homodimers are critical for plasmacytoid dendritic cell development [7, 8]. E2A also serves as an important dimerization partner for tissue specific HLH transcription factors outside of the lymphoid system. For example, E2A forms dimers with NeuroD and MyoD, key regulators of neuronal and skeletal muscle development, respectively [9–11]. Since E2A is the common factor for multiple lineage specific HLH transcription factor dimers, novel tools for targeting and manipulating E2A function can benefit a range of developmental biology research areas.
Here we have developed a new genetic model to examine E2A function. We have established an inducible E2A mouse model by inserting a tamoxifen-responsive region of the estrogen receptor (ER) ligand binding domain at the carboxyl end of tcfe2a, resulting in the production of E2AER fusion proteins. The use of tamoxifen inducible ER fusion proteins in mouse genetics has already been established as a valuable tool, especially with the vast use of the Cre recombinase-ER fusion protein for inducible gene knockout in mice [12–15]. In addition, ER fusion with a variety of transcription factors has also been successfully employed for analysis of gene expression. For example, MyoD-ER fusion proteins have been expressed by viral transduction in mouse embryonic fibroblasts for in vitro study of MyoD gene regulation  and in mouse fibroblasts for analysis of MyoD activation in vivo post transplantation of transduced cells into recipient animals . However, we do not know how useful the ER system will be in live animals when targeting an endogenous locus. Our E2AER system now introduces an ER fusion with a bHLH transcription factor into the mouse genome for analysis of an endogenously expressed protein.
E2AER/+ and E2AER/ER mice, along with E2AER protein function, are analyzed here in the context of B cell development. A block in B cell development at the pre-proB cell stage, prior to B lineage commitment, has been characterized by previous E2A-deficient mouse models [18, 19]. A rescue in B cell development from E2AER/ER pre-proB cells upon tamoxifen treatment would be a stringent test to verify inducible E2AER function.
In this study, we provide the initial characterization of the E2AER system. E2AER protein activity was rapidly induced upon tamoxifen treatment and reversibly regulated by tamoxifen withdrawal. Even though we show effective induction of E2AER DNA binding activity, tamoxifen treatment of E2AER/ER mice did not efficiently restore B cells in vivo. However, tamoxifen treatment was able to rescue and support early B cell development from E2AER/ER pre-proB cells in an ex vivo culture system. Use of E2AER ex vivo culture systems may therefore be beneficial for the study of gene regulation in B cells and other E2A-regulated cell lineages.
Generation of the E2AERallele
E2AER/+ and E2AER/ER mice were generated by using a knock-in strategy for tamoxifen-inducible E2A function. The tamoxifen-responsive ligand binding domain of the mouse ER  was inserted at the carboxyl end of tcfe2a to produce the E2AER allele (Figure 1A, B). With this targeting strategy, similar to that used for the E2AGFP strain previously developed in our lab , both alternatively spliced products of the tcfe2a gene, E12 and E47, are translated as ER fusion proteins. Initial characterization of the E2AER allele indicated normal expression levels of E2A mRNA in the presence of the ER insertion (Figure 1C). Previous study of E2A knockout mice has demonstrated stunted growth and a high lethality rate of homozygous animals within the first few weeks after birth [18, 19]. In contrast, the E2AE47bm strain, expressing a dominant negative form of E47, was originally described as indistinguishable from wild-type litter mates in size and survival . However, this work was analyzing mice on a mixed genetic background. Upon backcrossing to C57BL/6, the E2AE47bm/E47bm mice became smaller in size and demonstrated the high lethality rate like that shown with the knockout animals (unpublished data). E2AER/ER animals also exhibit stunted growth and reduced survival (see Additional file 1). Fortunately, the lethality rate in our experience has been less severe in litters from the E2AER strain than that observed with our E2A knockout and dominant negative strains. However, we do not know if this slight increase in postnatal survival is due to the presence of the E2AER protein or because our E2AER strain is currently on a mixed background.
The E2AERallele resembles an E2A-deficient allele in the absence of ligand
B cells develop from hematopoietic stem cells (HSC) in the bone marrow through a series of developmental stages . The pre-proB cell stage is an intermediate stage as lymphoid progenitors develop into committed proB cells. Pre-proB cells can be characterized by the expression of B220 and CD43 and the absence of CD19 expression. As pre-proB cells transition to the proB cell stage, CD19 expression is induced and cells undergo commitment to the B cell lineage. E2A is critical for this transition, as demonstrated by the block in development at the pre-proB cell stage in E2A-deficient animals [18, 19, 24]. Analysis of E2AGFP mice displays the up-regulation of E2A protein levels from the pre-proB to proB stage (see Additional file 2) . This increase in E2A expression is likely critical for E2A's regulation of the B cell lineage gene expression profile given the importance of E2A gene dosage. For example, elimination of one copy of E2A has been shown to greatly reduce the number of proB cells [4, 18].
Investigation of B cell development in E2AER/ER mice revealed a block at the pre-proB cell stage, similar to that seen in the E2A-mutant E2AE47bm/E47bm mice (Figure 2A) . Occasionally we have observed a small population of CD19+ B cells in the bone marrow of E2AER/ER mice, but these incidences of leaky B cell development did not appear to produce a significant population of mature, Ig expressing B cells (Figure 2B). Even though E2A has been suggested to influence proliferation in developing B cells [20, 25–27], no significant difference in expansion at the pre-proB cell stage was observed by BrdU analysis of E2AER/ER and wild-type mice (see Additional file 3).
For analysis of E2AER protein expression and DNA binding activity, we chose to derive Abelson transformed preB cells from E2AER/+ and E2AER/ER bone marrow . E2AER protein expression was verified by Western Blot analysis of whole cell lysates from E2AER/+ and E2AER/ER Abelson cells (Figure 3A). While E2AER protein levels appeared similar to wild-type levels when comparing E2A+/+ and E2AER/ER cells, analysis of E2AER/+ cells suggested that E2AER protein expression may be lower than wild-type. Protein analysis of tamoxifen treated cells displayed similar results, indicating that the relative E2AER protein levels are not affected by the presence or absence of tamoxifen. Nuclear extracts from E2AER/ER Abelson cells were then used to conduct electrophoretic gel shift analysis of E2AER binding to an E2A binding sequence, μE5. In the absence of tamoxifen treatment, no DNA binding activity was observed from the E2AER protein (Figure 3B). Together, the block in B cell development and lack of E2AER DNA binding activity suggest that the E2AER mouse model functions as an E2A-deficient system in the absence of ligand.
Rapid activation and reversible regulation of E2AER activity
Tamoxifen treatment of E2AER/ER Abelson cells resulted in rapid E2AER DNA binding activity within 1 hr of treatment (Figure 3B). The specificity of the protein binding to the μE5 probe was verified by using an anti-E2A antibody that effectively super-shifted the protein/DNA complex. The effect of tamoxifen withdrawal was then tested by washing tamoxifen-treated cells and growing them in the absence of tamoxifen for 1 and 6 hr time points. Loss of E2AER DNA binding activity was seen within 6 hrs of tamoxifen withdrawal (Figure 3C), indicating relatively fast reversibility of E2A function. These results demonstrate that the E2AER model can be used not only as an inducible model, but this system may also be valuable for providing a tightly regulated window of E2A activity.
Induction of E2AER activity supports early B cell development ex vivo
We first tested for a functional outcome of E2A induction by in vivo tamoxifen treatment of E2AER/ER mice followed by analysis of B cell populations in the bone marrow and spleen. All in vivo treatment efforts, including intraperitoneal injection and treatment in drinking water, unfortunately resulted in low efficiency rescue of B cell development. The emergence of B cells in tamoxifen-treated animals was rarely great enough to determine if the resulting B cells were generated in response to the tamoxifen treatment or were simply the incidence of leaky B cell development described above. The most significant recovery of B cells observed from in vivo treatment, which resulted from a 27 day tamoxifen treatment, was still considerably less than the B cell population in control mice (Figure 4A, B). However, the presence of Ig μ heavy chain (IgM) positive B cells in the spleen does suggest that a low level of tamoxifen-dependent B cell development occurred in vivo.
In contrast to in vivo tamoxifen treatment, treatment in an ex vivo B cell culture system effectively rescued the development of CD19+ B cells. Sorted E2AER/ER pre-proB cells were cultured in hormone-free media on an S17 stromal layer in the presence of IL-7, with or without tamoxifen. Control DMSO treated E2AER/ER pre-proB cells failed to develop efficiently into CD19+ B cells over the course of 5 days, whereas tamoxifen treated E2AER/ER pre-proB cells effectively gave rise to CD19+ B cells (Figure 5A). Interestingly, the kinetics of B cell development from tamoxifen treated E2AER/ER pre-proB cells appeared delayed compared to that of control cells. In addition to using CD19 expression to validate the rescue of B cell development, we analyzed Pax5 expression throughout the 5 day culture. Pax5, initiated downstream of E2A expression, is a transcription factor critical for B cell lineage commitment . Consistent with CD19 expression, induction of Pax5 expression was observed in tamoxifen treated E2AER/ER pre-proB cells, also appearing delayed compared to control cultures (Figure 5B).
Another key event during early B cell development, downstream of E2A function, is rearrangement of the Ig heavy chain locus (IgH) [2, 18, 19]. We next used this culture system to determine if induction of E2AER activity could also support IgH V to DJ recombination. To amplify V to DJ rearrangements, we chose primers recognizing members of the VH1 gene family , which represent a large percentage of the total IgH V genes, and primers recognizing the JH4 gene segment. Rearrangements were analyzed in Day 8 cultured samples because we did not observe consistent V to DJ PCR signals until this point in the time course analysis. Wild-type mice were used as positive controls for detecting recombination events, and mice deficient in the recombination activating gene RAG1, required for V(D)J recombination, were used as negative controls. Analysis of Day 8 cultures demonstrated a clear V to DJ rearrangement product from tamoxifen treated E2AER/ER cells (Figure 6). V to DJ rearrangement was further verified by sequencing analysis (see Additional file 4). The faint product detected from DMSO control treated E2AER/ER cells was accompanied by a small "leaky" CD19+ population generated at this relatively late time point in the culture system (see Additional file 5). However, only one unique rearrangement product was identified out of all of the colonies sequenced for this sample. In addition to providing functional proof of induced E2A activity, the detection of IgH recombination, along with the induction of CD19 and Pax5 expression described above, suggests that restored E2A function can rescue B cell development from the pre-proB cell stage.
The E2AER system provides an improved method for analyzing E2A function. Previous analysis using over-expression of E2AER fusion proteins by retroviral transduction has already demonstrated the value of inducible E2A activity [20, 31–34]. However, toxicity is often a problem with high levels of E2A in the cell [20, 35], and viral transduction is not ideal for all cell types. In addition, the changes in gene expression detected upon over-expression of E2A may not always be representative of endogenous E2A function. Therefore, generation of the E2AER allele provides an attractive system for studying gene regulation and other E2A-regulated events in potentially any E2A-expressing cell type at a more physiological level.
Here we demonstrate the rapid induction of E2AER DNA binding activity upon tamoxifen treatment and its potential for reversible function upon tamoxifen withdrawal. The ability to tightly control E2A activity allows for kinetic analysis of downstream events. Analysis of B cell development in our ex vivo culture system demonstrated a rescue from the pre-proB cell stage, but suggested delayed kinetics from E2AER/ER pre-proB cells compared to B cell development from control pre-proB cells. This result suggests that even though E2AER/ER and wild-type pre-proB cells are phenotypically similar, E2AER/ER pre-proB cells may not have equivalent developmental potential or may require additional cellular changes prior to entering the B cell lineage. It is possible that E2A-regulated events normally occurring earlier in development, as suggested in hematopoietic stem cell (HSC), lymphoid-primed multipotent progenitor (LMPP), and common lymphoid progenitor (CLP) stages [27, 36, 37], were occurring upon restored E2A function in pre-proB cells and requiring these cells to "catch up" with their wild-type counterparts prior to progressing to the next stage. Alternatively, the developing tamoxifen-treated E2AER/ER B cells may not have expanded as rapidly as wild-type cells upon commitment to the B cell lineage. Our Western Blot analysis of E2A proteins in E2AER/+ Abelson cells (Figure 3A) suggested that the E2AER protein level may also have been contributing to the delayed kinetics. In this sample, the E2AER band appeared less intense than the wild-type E2A band. However, it is not possible to determine from this analysis if the lower intensity was a result of reduced protein levels or variation in antibody affinity for the E2AER versus wild-type E2A protein. In addition, there was not a striking difference between the E2AER and wild-type protein levels when comparing the E2A+/+ and E2AER/ER samples. Further investigation of tamoxifen treated E2AER/ER pre-proB cells will be necessary to understand why the delayed detection of CD19+ B cells was observed.
Our ex vivo culture data clearly indicated a rescue of early B cell development to the proB cell stage. However, we have so far been unable to determine if tamoxifen treatment can rescue E2AER/ER B cells through the subsequent preB cell stage, when E2A is known to be critical for Ig kappa light chain (Igk) recombination . We were unable to test for induction of Igk recombination or surface IgM expression by using our ex vivo culture system because this system primarily supports development at a stage prior to these events (see Additional file 5 and data not shown). The presence of the small, but increased, population of E2AER/ER IgM+ B cells in the in vivo treatment experiment (Figure 4A) is the only current evidence suggesting tamoxifen-induced E2AER activity may be able to rescue B cell development to maturity. Additional B cell culture systems would be necessary to determine if tamoxifen treatment can rescue later stages of E2AER/ER B cell development.
While in vivo tamoxifen treatment was not efficient for restoring B cell development, in vivo treatment may still be useful for analysis of E2A function in other cell lineages. We suggest two reasons for ineffective rescue of in vivo B cell development. First, progression through several stages of lymphocyte development is known to be dependent on proper E-protein dosage [4, 6, 18, 26, 39, 40], and it is possible that the required E2A threshold is not maintained during our in vivo treatment for rescue of B cell development. For example, as mentioned above, we do not know if E2AER/ER cells can sufficiently progress through the preB cell stage of development. Second, E2A has been suggested to be important for B cell survival . Therefore, it is conceivable that even if B cells are rescued by tamoxifen treatment, they may be lost if E2AER activity is not maintained throughout the treatment.
The E2AER system may instead be more valuable for in vivo study of T cell development, or other lineages expressing multiple E-protein family members. Since E-proteins demonstrate many redundant functions, T cell development is not completely blocked in E2A-deficient mice due to the presence of HEB [4, 6, 42, 43]. Because T cells are fully developed in E2A-deficient mice, in vivo tamoxifen treatment of E2AER/ER mice may be more valuable for the study of E2A function during T cell development.
There are many additional applications for the E2AER system. In addition to advantages in speed, expression level, and reversibility, this inducible system may allow analysis of E2A function in specific cell stages that have been difficult to analyze in previous E2A-deficient models. Tamoxifen treatment of E2AER/ER cells may be valuable by allowing progression to developmental stages that are absent in E2A-deficient animals or affected due to the lack of E2A at earlier developmental stages. E2AER/ER mice also provide a useful tool for studying changes in E2A function with age or for analyzing E-protein function in combination with other genetic models. Given the rapid induction of E2AER DNA binding activity upon in vitro tamoxifen treatment, we believe that the use of E2AER ex vivo culture systems may be most valuable, especially for identifying new E2A targets and studying the kinetics of E2A gene regulation.
We have established a new genetic model by generating the E2AER allele. This model allows for inducible function of E2A, a transcription factor displaying an extensive range of functions across multiple developmental programs. E2AER activity is rapidly induced at the protein level upon tamoxifen treatment and is reversible upon tamoxifen withdrawal. Tamoxifen treatment of E2AER/ER mice was, however, unable to efficiently restore wild-type levels of B cells. The functionality of E2AER was instead successfully verified by ex vivo tamoxifen treatment of E2AER/ER B cell progenitors. In this ex vivo culture system, induced E2AER protein function was able to rescue and support early B cell development. Thus, the E2AER model provides an attractive system to regulate and study E2A protein function, especially under ex vivo conditions where cells can gain sustained access to high levels of tamoxifen.
E2AE47bm mice have been described previously . Generation of the E2AER allele is described below. All research with mice was performed in accordance with relevant guidelines, and protocols were approved by the Duke University Animal Care and Use Committee.
Generation of the E2AER allele
The gene targeting strategy used was a modification of the strategy for generation of the E2AGFP strain . The tamoxifen-responsive region of the mouse estrogen receptor ligand binding domain containing the G525R mutation  was PCR amplified from the MigR1-E47R vector  using the primers ERfpA: 5'-CGGATCCACGAAATGAAATGGGTGC-3' and ERrpA: 5'-CCGGCCGCTAGAATTCGATCGTGTTGGGGAAGCCCTC-3' to introduce a 5' BamHI site and 3' EcoRI and EagI sites for subsequent cloning steps. The ER fragment was inserted, replacing EGFP, at the BamHI position in frame with E2A. The targeting construct also contained a positive selection marker, PGKNeo cassette, and a negative selection marker, PGK driven thymidine kinase (TK) cassette. Mouse ES cells used were derived from a 129/sv strain obtained from Phillippe Soriano's lab in 1995 and then maintained in our own lab. E2AER/+ and E2AER/ER mice were maintained on a C57BL6 and 129/sv mixed background. Three primers were used for detection of wild-type and mutant alleles, yz164: 5'-AAGAACGAGGCCTTCCGTGTC-3', yz29: 5'-TCGCAGCGCATCGCCTTCTA-3', and bjE2Ar3: 5'-CAAGAGACTAGGATGCCACTG-3'.
RNA extraction, DNase I treatment and reverse transcription have been described previously . Quantitative real-time PCR analysis for Pax5 expression was performed using a Roche LightCycler and Fast-Start DNA master SYBR green kit I (Roche) as per manufacturer's instructions. The following primers were used, E2A: f1 5'-CCAGTCTCAGAGAATGGCAC-3' and r1 5'-CCTTCGCTGTATGTCCGGCTAG-3'; Pax5 and GAPDH primers .
Cell staining and flow-cytometry
For sorting, bone marrow was harvested and pooled from 2-3 mice per genotype. Cells positive for lineage markers Mac-1, Gr-1, Ter-119, and CD3 were depleted with Dynal Dynabeads (Invitrogen) according to manufacturer's instructions. Dead cells stained with 7-aminoactinomycin D (7AAD, Molecular Probes) were excluded. Pre-proB cells were further distinguished as B220+CD43+CD19-. FACS analysis was done with a FACSCalibur (BD Biosciences) or FACSVantage SE with DiVa option (BD Biosciences) and FlowJo software (Tree Star). FACSVantage SE with DiVa option was used for cell sorting.
Tamoxifen preparation for in vitroculture treatment
Tamoxifen (Sigma) was prepared as a 1 mM stock (1000×) dissolved in cell culture grade dimethyl sulfoxide (DMSO) and stored at -20°C.
Abelson transformed preB cells
The E2AER/ER Abelson preB cell line was derived by Abelson Murine Leukemia Virus transformation of bone marrow cells from an E2AER/ER mouse. Briefly, whole bone marrow was plated on an S17 stromal layer in the presence of 1 uM tamoxifen and 10 ng/mL IL-7 in 5% FBS RMPI media. This culture was performed prior to transduction to ensure cells were proliferating and were at the optimal target stage for Abelson transformation. Once an expanding B cell population was observed, cells were infected with Abelson virus in the presence of 4 ug/mL polybrene. Abelson transformed cells were then removed from the stromal layer, and tamoxifen and IL-7 were withdrawn. The established E2AER/ER Abelson preB cell line was maintained in 10% FBS RPMI media (also containing 100 units/ml penicillin, 100 ug/mL streptomycin and 55 uM 2-mercaptoethanol) prior to experimental analysis. Additional Abelson lines were established as described previously .
Abelson preB cell lines were cultured without tamoxifen or with 1 uM tamoxifen for 24 hr prior to analysis. Cells were lysed in RIPA lysis buffer (1% Triton, 0.5% sodium deoxycholic acid, 0.1% SDS, 25 mM Tris-Cl pH 7.6, 150 mM NaCl, 5 mM EDTA) with protease inhibitors. Whole cell lysates were resolved by SDS-PAGE and blotted with anti-E2A (G127-32, BD Biosciences, 554077) and anti-ERK2 (C-14, Santa Cruz Biotechnology, sc-154) antibodies.
Electrophoretic mobility shift assay
E2AER/ER Abelson preB cells were cultured with or without 1 uM tamoxifen as indicated. For withdrawal analysis, tamoxifen-treated cells were washed and re-plated in the absence of tamoxifen for the indicated times. Nuclear extracts were incubated with a 32P-labeled μE5 oligonucleotide probe, with or without Yae anti-E2A monoclonal antibody (Santa Cruz Biotechnology, sc-416), and resolved on a 5% polyacrylamide gel. Gels were dried and exposed to a phosphor screen for phosphorimager analysis (Amersham Biosciences). Oligos used for μE5 probe: 5'-TCGAAGAACACCTGCAGCAGCT-3' and 5'-TAGAGCTGCTGCAGGTGTTCTT-3'.
In vivotamoxifen treatment
Mice were treated with tamoxifen in the drinking water for 27 days. A 68 mg/mL tamoxifen in ethanol stock was used to bring the concentration in drinking water to approximately 26 ug/mL, resulting in 0.04% ethanol in water. A fresh bottle of tamoxifen water was given every 5 days.
Ex vivopre-proB culture system
Sorted pre-proB cells were plated on an S17 stromal layer in 24-well plates at approximately 1.5 × 104 cells per well and cultured with 5% FBS RPMI hormone-free media containing 10 ng/mL IL-7. Hormone free media consisted of phenol-red free RPMI 1640 supplemented with 5% charcoal/dextran treated FBS (Hyclone), 100 units/mL penicillin, 100 ug/mL streptomycin, and 55 uM 2-mercaptoethanol. Treated wells contained 1 uM tamoxifen and untreated controls were given DMSO alone (0.1%). Cells received fresh media, cytokine, and tamoxifen or DMSO every other day. Cells were harvested at time points indicated, and samples were split in half for FACS analysis and RNA or DNA extraction.
IgH rearrangement analysis
Sorted pre-proB cells were ex vivo cultured as described above. DNA was extracted from Day 8 cultures and then analyzed for IgH V to DJ rearrangements using VH1 gene family and JH4 specific primers. A nested PCR strategy was used to amplify rearrangements involving VH1 family gene segments. The following primers were used for Round 1: VH1 ext 5'-AGRTYCAGCTGCARCAGTCT-3'  and JH4 YZB6 5'-TCCCTCAAATGAGCCTCCAAAGTCC-3'  and for Round 2: VH1 int 5'-GARGATRTCCTGYAAGGCTTC -3'  and JH4 YZB5 5'-CCTGAGGAGACGGTGACTGAGGTTCCTTG-3'. CD14 primers were used to demonstrate the presence of DNA in all samples . VH1-DHJH4 PCR products were cloned into a pCR4 TOPO vector (Invitrogen) and sequenced (Direct Sequencing from Colonies Service, Eton Bioscience, Inc). Rearrangement product sequences were analyzed by SoDA .
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We thank the Duke University transgenic facility for assistance with generating the knock-in mice, the Duke Comprehensive Cancer Center Flow Cytometry facility for help with cell sorting, Weiguo Zhang's lab for assistance with Western Blot analysis, and Adam Lazorchak for helpful discussions and technical support for EMSA analysis. This work was supported by funding from the National Institutes of Health to YZ.
MEJ participated in the design of the study, carried out the studies, and drafted the manuscript. MK participated in the design of the study and assisted with cell sorting. YZ conceived of the study and participated in its design. All authors read and approved the final manuscript.