- Methodology article
- Open Access
LoxP-FRT Trap (LOFT): a simple and flexible system for conventional and reversible gene targeting
- Barbara H Chaiyachati†1,
- Ravinder K Kaundal†1,
- Jiugang Zhao1, 2,
- Jie Wu1,
- Richard Flavell1 and
- Tian Chi1Email author
© Chaiyachati et al; licensee BioMed Central Ltd. 2012
- Received: 15 October 2012
- Accepted: 30 November 2012
- Published: 30 November 2012
Conditional gene knockout (cKO) mediated by the Cre/LoxP system is indispensable for exploring gene functions in mice. However, a major limitation of this method is that gene KO is not reversible. A number of methods have been developed to overcome this, but each method has its own limitations.
We describe a simple method we have named LOFT [LoxP-flippase (FLP) recognition target (FRT) Trap], which is capable of reversible cKO and free of the limitations associated with existing techniques. This method involves two alleles of a target gene: a standard floxed allele, and a multi-functional allele bearing an FRT-flanked gene-trap cassette, which inactivates the target gene while reporting its expression with green fluorescent protein (GFP); the trapped allele is thus a null and GFP reporter by default, but is convertible into a wild-type allele. The floxed and trapped alleles can typically be generated using a single construct bearing a gene-trap cassette doubly flanked by LoxP and FRT sites, and can be used independently to achieve conditional and constitutive gene KO, respectively. More importantly, in mice bearing both alleles and also expressing the Cre and FLP recombinases, sequential function of the two enzymes should lead to deletion of the target gene, followed by restoration of its expression, thus achieving reversible cKO. LOFT should be generally applicable to mouse genes, including the growing numbers of genes already floxed; in the latter case, only the trapped alleles need to be generated to confer reversibility to the pre-existing cKO models. LOFT has other applications, including the creation and reversal of hypomorphic mutations. In this study we proved the principle of LOFT in the context of T-cell development, at a hypomorphic allele of Baf57/Smarce1 encoding a subunit of the chromatin-remodeling Brg/Brahma-associated factor (BAF) complex. Interestingly, the FLP used in the current work caused efficient reversal in peripheral T cells but not thymocytes, which is advantageous for studying developmental epigenetic programming of T-cell functions, a fundamental issue in immunology.
LOFT combines well-established basic genetic methods into a simple and reliable method for reversible gene targeting, with the flexibility of achieving traditional constitutive and conditional KO.
- Green Fluorescent Protein
- Green Fluorescent Protein Expression
- LoxP Site
- Floxed Allele
- High Mobility Group Domain
Conventional gene knockout (KO) technologies such as LoxP/Cre-mediated conditional gene KO (cKO) are widely used for discovering gene functions. A key limitation of these methods is that the KO is irreversible. It is therefore impossible to determine if, for example, the malignancies and neurological disorders reported in p53 and MeCP2 KO mice, respectively, can be cured by restoring gene functions, a question of obvious clinical relevance. Because the KO in the original mouse models is not reversible, special strains have to be generated to address these questions, which entails substantial amounts of work [1–3]. Reversible KO would also be invaluable for studying epigenetic programming, a central issue in developmental biology. Specifically, during lineage development, transient action of environmental cues is thought to irreversibly modify (or 'program') the epigenetic states of target genes in the developing cells, such that the altered epigenetic states can persist and be propagated to mature progeny cells without the continuous presence of the initiating cues . Defining the role of a gene in developmental programming requires deleting the gene in immature cells and analyzing the resultant defects in mature cells, but the gene controlling developmental programming may also be expressed and functioning in mature cells, which complicates data interpretation, given that conventional KO strategy is not reversible. For example, deleting the chromatin-remodeling factor Mi-2b in immature T cells impairs proliferation of mature T cells , but because Mi-2b is expressed not only in immature but also in mature T cells, it is unclear if the proliferation defect reflects a developmental role of Mi-2b. The only way to directly address such an issue is to eliminate the protein in immature cells. and then restore its expression in mature cells.
Multiple methods have been devised to achieve reversible gene regulation, but each has limitations. In one method, endogenous genes are modified so that their expression is now driven by tetracycline-regulated artificial transcription activators expressed from the endogenous regulatory elements, thus allowing for reversible gene regulation, but it is difficult to recapitulate the expression levels of the endogenous genes with the synthetic activators [6–9]. In an alternative method, tetracycline-controlled transcriptional silencer (tTS), a tetracycline-regulated transcription repressor, has been successfully used to reversibly inhibit the expression of Hoxa2 and Htr1a, but whether this method is generally applicable to other genes remains unclear, and furthermore, the only tTS transgenic line currently available expresses tTS in various tissues, and is hence unsuitable for tissue-specific inhibition. Regulated expression of small hairpin RNA has also been used for reversible gene repression, but the repression is usually incomplete . Finally, transcription stop sequences or gene-trap cassettes, which are removable/inactivable, can be inserted into target genes, leading to constitutive KO that can be conditionally rescued, but this strategy is not suitable for conditional induction of gene KO [1, 3, 10, 11].
In this paper, we describe a straightforward and robust method for reversible cKO without these limitations. The method, which we dub LOFT [LoxP-flippase (FLP) recognition target (FRT) Trap], combines cKO with gene trapping, a well-established method for insertional mutagenesis [12–16]. In its simplest form, a gene-trap cassette consists of a promoterless selectable marker flanked by a splice acceptor (SA) and a polyadenylation (pA) sequence. When inserted into an intron of an expressed gene, the SA captures the upstream exon while the pA sequence truncates the transcript, thus producing a fusion protein between the N-terminus of the trapped protein and the selectable marker. Thus, gene traps simultaneously inactivate and report the expression of the trapped gene. Gene trapping can be made conditional by flanking gene-trap modules with LoxP/FRT sites [10, 11, 17]. LOFT combines Cre-catalyzed cKO with FLP-catalyzed reversible trapping to achieve reversible cKO. LOFT can also be used to create conventional KO mice. We report a proof-of-concept study using the gene encoding Brg/Brahma-associated factor (BAF)57, a subunit of the chromatin-remodeling BAF complex.
The BAF complex, a prototypical mammalian ATP-dependent chromatin remodeler complex (CRC), is widely expressed, and plays diverse, often tissue-specific roles in gene regulation [18–20]. Although called ATP-dependent CRC, the complex can also regulate target genes without using the classic ATP-dependent chromatin-remodeling activity . Indeed, although the complex consists of more than ten subunits, a group of four core subunits, including the catalytic subunit Brahma-related gene (BRG)1, is fully sufficient to reconstitute ATP-dependent chromatin-remodeling in vitro . The functions of the remaining accessory subunits are poorly understood, but may contribute to the ATP-independent functions of the BAF complex and/or modulate the classic remodeling activity of the BAF complex. The 57 kDa high mobility group (HMG) protein BAF57 (also known as SMARCE1; Switch/sucrose non-fermentable (SWI/SWF) related matrix-associated actin-dependent regulator of chromatin subfamily E member 1) is the first known accessory subunit . BAF57 is important for T -cell development in mice , and for regulating apoptosis , the cell cycle  and functions of the androgen and estrogen receptors [27–29] in tumor lines. Furthermore, BAF57 is strongly expressed in human endometrial carcinoma, and serves as a marker of poor prognosis .
We are interested in further studying the roles of BAF57 in T- cell development in the thymus, which is arguably the best-defined ontogenetic system in vertebrates . The earliest thymocytes are double-negative (DN) cells lacking the antigen coreceptor CD4 or CD8. These cells undergo extensive proliferation, and express both CD4 and CD8 to become double-positive (DP) cells. DP cells bifurcate into CD4 helper and CD8 cytotoxic cells, the two major subsets of T lymphocytes in the adaptive immune system, which are marked by CD4 and CD8 expression, respectively. We previously explored the role of BAF57 in T cells using a BAF57 dominant-negative mutant. BAF57 is a protein of 411 amino acids (aa) consisting of several conserved domains, including the N-terminal proline-rich domain (23 aa) with unknown functions, the HMG domain (aa 66 to 133) that binds DNA, a domain rich in Asp, His, Leu and Ile (NHRKI), and the C-terminal domain rich in acidic residues . The functions of these domains are unknown except for the DNA-binding of the HMG domain . We found that thymocyte-specific expression of a dominant-negative mutant of BAF57 lacking the N-terminal 133 aa including the HMG causes reciprocal CD4/CD8 misregulation during T-cell development, but the mutant does not significantly impair production or function of mature T cells [24, 33]. Because the dominant-negative mutation impairs only a specific aspect of BAF57 function, the roles of BAF57 in T cells remain incompletely understood. In particular, it is unclear if BAF57, acting in thymocytes, can epigenetically program the function of mature T cells. This problem motivated us to develop the reversible cKO method LOFT.
LOFT: Basic rationale
Generation of Baf57 F and Baf57 ΔR
Baf57 F and Baf57 Δ R were generated with a single targeting vector by exploiting crossing-over site variability during homologous recombination (Figure 2) [35–37]. To facilitate the generation of Baf57 Δ R lacking the 5' LoxP site, we used a short (0.5 kb) left arm upstream of the 5' LoxP site. This was used to ensure that during homologous recombination between the left arm and the endogenous sequence, the crossing over could take place not only upstream of the 5' LoxP site, leading to its incorporation into the endogenous gene, but also downstream of the LoxP site, preventing its incorporation. The targeted allele retaining the 5' LoxP site (Baf57 Int ) can be converted to Baf57 F after FLP-mediated excision of the gene-trap cassette from the germline, whereas the allele lacking the 5' LoxP site is Cre-resistant and acts as Baf57 ΔR .
Characterization of Baf57 F and Baf57 ΔR
To determine whether Baf57 ΔR could be conditionally activated through deletion of the gene-trap cassette, we used the R26 FlpoER1 deleter strain that ubiquitously expresses FlpoER1 from the Rosa26 locus . FlpoER1 is a fusion between the codon-optimized FLP called FLPo [41, 42] and the modified estrogen receptor (ER), ERT2, which retains the recombinase in the cytosol until tamoxifen (TAM) administration , with a linker sequence derived from Cre-ER inserted between the FLPo and ERT2. In mice carrying Baf57 ΔR and expressing FlpoER1, TAM injection therefore would induce nuclear translocation of FlpoER1 to cause deletion of the gene-trap cassette and hence activation of the Baf57 ΔR . To determine the ability of FlpoER1 to activate Baf57 ΔR , we introduced R26 FlpoER1 into Baf57 F/ΔR mice. We injected TAM once a day for 3 consecutive days, and monitored GFP expression in peripheral blood lymphocytes. Before TAM injection, GFP was uniformly expressed in CD4, CD8 and B cells (Figure 4C, top middle panel). Cells losing GFP emerged 6 days after the first TAM injection, and comprised around 50% of total lymphocytes on day 10, when the GFP signals in the affected cells were reduced by about 4-fold (Figure 4C, bottom middle plot). Because the GFP half-life is ~24 hours and the peripheral lymphocytes are mostly resting, deletion of the gene-trap cassette in our mice seemed to occur predominantly around day 8 after the first TAM injection. A similar observation was made in a Baf57 F/ΔR ; CD4-Cre; R26 FlpoER1 mouse, except that on day 10, only about 30% of the lymphocytes had deleted the gene-trap cassette (Figure 4C, right). The variation in the deletion efficiency was stochastic and not correlated with genotype; on average, the deletion efficiencies at day 10 were 37 ± 8%, 37 ± 7%, and 51 ± 8% in CD4, CD8, and B lymphocytes, respectively (Figure 4D). We sorted GFP+ and GFPlow/- cells from Baf57 F/ΔR ; CD4-Cre; R26 FlpoER1 mice, and performed western blotting. As expected, the GFP+ cells expressed only the three truncation mutants and no WT BAF57 protein (Figure 4A, lane 9), and importantly, WT BAF57 was restored in GFPlow/- cells, showing that Baf57 ΔR can indeed be converted into the WT allele (Figure 4A, lane 8).
We next sought to determine the identities of the three mutants expressed by Baf57 F after Cre-mediated deletion of exons 2 to 3 (these mutant referred to as Baf57 Δ(2-3) hereafter). As mentioned before, Baf57 Δ(2-3) is expected to direct the expression of the BAF57 mutant that lacks the first 18 residues and hence is about 2 kDa smaller than the WT protein, which might account for one of these three mutants, as the molecular weights of these mutants seemed to differ from the WT protein by less than 5 kDa. The other two mutants might be its degradation products, and/or be expressed from aberrantly spliced transcripts. To address this, we performed reverse transcriptase (RT)-PCR using primers targeting exons 1 and 7. The primers amplified a single product of 839 bp in WT CD4 cells as expected, but produced two smaller bands from Baf57 F/ΔR ; CD4-Cre mice (Figure 4B, lanes 1 and 2). RT-PCR using primers targeting exons 1 and 5 suggested that the top and bottom bands in the mutant cells represented the predicted transcript (with exon 1 joined to exon 4) and an aberrant transcript with exon 1 joined to exon 5, respectively (Figure 4B, lanes 3 and 4), which was confirmed by restriction enzyme digestion of the amplicons (lanes 5 to 10). Interestingly, exon 5 was also found to harbor an in-frame ATG, suggesting that the aberrantly spliced transcript can be translated into a deletion mutant lacking the N-terminal 70 aa (including the first 5 aa of the HMG domain) and hence is about 8 kDa smaller than WT protein. Perhaps this mutant was running aberrantly slowly to constitute one of three mutant bands. Of note, as in the case of the ATG in exon 4, the ATG in exon 5 is not embedded in the Kozak consensus sequence, consistent with their low expression levels compared with the WT protein. Thus, at least two of the three mutant proteins might result from translation of spliced transcripts. Indeed, multiple alternatively spliced transcripts, two of them predicted to direct the expression of the mutant proteins lacking the N-terminal 18 and 70 aa, normally exist in the brain . However, direct sequencing of these bands is needed to confirm this hypothesis, particularly because of the unusual mobility of BAF57, whose predicted molecular weight is 45 kDa but whose apparent molecular weight ranges from 50 to 57 kDa, depending on the gel system used.
Finally, as alluded to before, although the three mutant proteins were expressed, as expected, only at very low levels in the cells that also expressed WT protein, they accumulated in the cells lacking the WT protein, which occurred both in the thymus (Figure 4A, lanes 2 versus 5) and the mature CD4 cells (Figure 4A, lanes 6 versus 7, and 8 versus 9). This upregulation of the mutant proteins in the absence of WT BAF57 presumably reflected a post-translational regulatory mechanism seeking to maintain stoichiometric abundance of various BAF subunits . As the mutant proteins are at least partially active, their accumulation may help explain why deletion of the floxed exons in Baf57 F had no major biological effect. However, because the mutants presumably lacked the intact N-terminus, their accumulation might recapitulate, to some extent, the phenotype seen in mice overexpressing a BAF57 dominant-negative mutant lacking the N-terminal 133 aa, and more importantly, this defect may be prevented by TAM treatment. This is indeed the case, as described below.
Phenotype caused by Baf57mutations and its prevention by TAM injection
The fact that Baf57 F/ΔR ; CD4-Cre mice showed a significant defect in CD8 expression set the stage for testing the effect of TAM on the phenotype. Given the rapidity of early T-cell development and the relatively slow kinetics of Baf57 ΔR activation, it is unfeasible for TAM to 'cure' the pre-existing phenotype in early DP cells that exist only transiently, but TAM might be able to activate Baf57 ΔR in the precursors of these transient cells to prevent the subsequent defective CD8 expression in early DP cells. To test this, we used Baf57 F/ΔR ; CD4-Cre; R26 FlpoER1 and Baf57 F/ΔR ; R26 FlpoER1 mice, the latter included because Baf57 ΔR alone sufficed to produce a detectable, albeit weak, defect in CD8 expression. The mice were exposed to TAM as described above. Unexpectedly, on day 10 after the initial TAM injection, only about 5% of the early thymocytes had lost GFP expression (Figure 5, rows F to G, column 1), and the same was true for later thymocytes (not shown), in contrast to the approximately 37% deletion efficiency in peripheral lymphocytes, thus revealing substantial tissue-specificity in the deletion efficiency in our system. We then examined early DP cells showing or lacking GFP expression. As expected, in cells expressing GFP, CD8 expression was impaired, with the CD4+CD8low/- population comprising about 10% and 25% of the early thymocytes in Baf57 F/ ; R26 FlpoER1 and Baf57 F/ΔR ; CD4-Cre; R26 FlpoER1 mice, respectively (Figure 5, rows F to G, columns 2). Importantly, the CD4+CD8low/- population was absent in the GFP- compartment, demonstrating successful prevention of the phenotype (Figure 5, rows F to G, column 3). This effect was dependent on R26 FlpoER1 (not shown) and thus not an artifact resulting from elimination of GFP (see Additional file 1, Figure S2).
Strengths and limitations of LOFT
LOFT combines pre-existing basic genetic methods into a straightforward and reliable reversible gene-targeting method. The method is reliable because its two components, Cre-mediated conditional gene targeting and FLP-mediated reversible gene trapping, are both well established. It is also simple because the pair of alleles involved can typically be generated with a single construct. Furthermore, for the genes whose floxed alleles are already available, only the trapped alleles are needed to convert the pre-existing cKO into reversible cKO, which simplifies the method. This is an important advantage because floxed alleles for the majority of mouse genes will become available in the future thanks to concerted efforts in several countries [46, 47]. LOFT is also flexible, because the two alleles can be used independently for conventional gene targeting, and what is more, the 'intermediate allele' generated can serve as a GFP reporter of Cre activities. LOFT does require Cre and FLP-deleter lines, but this should not pose a problem because numerous Cre lines are already available, as is a mouse line ubiquitously expressing a version of FlpoER that is far more effective than the one (FlpoER1) used in the current study; the two versions are identical except for the linker sequence between FLPo and ER . Finally, LOFT, whose major application is likely to be reversible cKO, can have other applications such as reversal of hypomorphic alleles, as we have shown. As another application, point mutations may be introduced into the trapped genes to dissect their functions, which is analogous to the approach for producing conditional point mutant mice that we previously developed , except that in the previous method, the point mutant is expressed concurrently with the loss of the WT protein, whereas in the LOFT method, the two events can happen sequentially. The sequential occurrence would be essential in addressing, for example,, the mode of action of P53. Specifically, KO of the P53 gene is known to cause tumors, which can be suppressed by restoration of P53 expression [1, 2]. Surprising, it was recently found that a P53 point mutant unable to induce apoptosis, cell-cycle arrest, or senescence retained the ability to prevent tumorigenesis, presumably as a result of the ability of the mutant protein to regulate energy metabolism and production of reactive oxygen species . Whether the mutant can also suppress pre-existing tumors is unclear, and this important question is readily addressable by expressing the mutant protein in P53 cKO mice. In summary, LOFT is a straightforward, reliable, simple, and flexible method for both reversible and conventional (constitutive or conditional) gene targeting, and is readily adaptable for other applications.
However, there are several limitations to LOFT. First, the method involves a pair of alleles with the corresponding pair of recombinases, thus entailing significant amounts of breeding. Second, the trapped allele is null by default, and so the gene needs to be haplosufficient for mouse survival. Third, the method is not suitable for reversing the effect of deletion of regulatory elements such as enhancers or silencers. Fourth, because LOFT works by combining Cre/Lox and gene-trapping systems, any limitation in these basic genetic methods would apply to LOFT. For example, as mentioned above, if the target gene is not expressed in ES cells, then 'targeted trapping' is not applicable, and conventional methods, which have much lower efficiency, must be used . Fortunately, over 65% of all protein-coding genes in the mouse genome are amenable for promoter trapping in ES cells , and the efficiency can be raised to 85% if the binding sites (which can be made removable) for a transcription factor expressed in ES cells (Oct4) are engineered into the vector . Thus, inefficient conventional methods should be reserved for only around 15 to 35% of protein-coding genes. Because conventional methods are well established, we do not expect unusual problems in their application to our setting. Another example of the limitation of LOFT is that if two targeting constructs are needed to insert the 5' and 3' LoxP sites, as in the case of floxing a large DNA fragment, then creating the allelic series in LOFT will accordingly require two constructs. Furthermore, Cre/FLPo cannot always efficiently delete target sequences. Indeed, FLPo-catalyzed removal of the gene-trap cassette at the BAF57 locus was only around 5% in the thymus by day 10. This problem can be addressed by monitoring the deletion via GFP expression, and by the use of the new version of conditional FLP ("FlpoER") that is efficient 'in any tissue at any time during development or in the adult' . With this version of FLP, Joyner and colleagues found that a single injection of TAM was sufficient to induce widespread and efficient deletion of a reporter gene in the embryos and adults within 4 and 7 days, respectively, whereas the version used in the current study (FlpoER1) barely works under this condition . The new enzyme is expected to make LOFT widely applicable. However, the old version (FlpoER1), which is efficient in peripheral T cells but not thymocytes, has a unique advantage for studying developmental programming of T-cell functions. As mentioned above, such studies entail gene inactivation in the thymocytes and subsequent reactivation in mature T cells. As the thymus continues to export T cells into the periphery in adults, the peripheral T-cell pool would be significantly contaminated with the confounding T cells that have undergone premature reactivation, if FLP is allowed to work efficiently in the thymocytes. The final limitation of LOFT involves the fact that most of the floxed alleles recently generated by the European Conditional Mouse Mutagenesis (EUCOMM) carry an FRT site outside the floxed sequence. If these alleles are paired with reversible KO alleles, interchromosomal recombination can occur after FLP activation. These recombination events are presumably too rare to confound data interpretation, unless they lead to dominant effects such as tumorigenesis. However, such effects are in themselves interesting, and so the nuisance may be a blessing in disguise.
Utility of Baf57 ΔR and Baf57 F
BAF57ΔR is a null allele that can be rescued by deleting the gene-trap cassette. BAF57ΔR homozygous mice are apparently embryonic lethal, which precludes the analysis of the effect of BAF57 KO in adult tissues. This problem may be solved by deleting the gene-trap cassette (and hence restoring BAF57 expression) in a fraction of cells in the embryo, which may rescue the embryo to produce mosaic adults containing BAF57 KO, Het, and WT cells, which are distinguishable based on (the level of) GFP expression. Such mosaic mice will be the source of cells lacking BAF57.
In contrast to Baf57 ΔR , Baf57 F was designed to be a conditional hypomorphic allele expressing a deletion mutant at a low level, following excision of the floxed sequence. Instead, we detected three truncated proteins that seemed to result, at least in part, from alternatively spliced transcripts. In addition, although the mutants were indeed expressed at very low levels in the presence of the allele expressing WT BAF57, they accumulated in the absence of WT BAF57. Despite these unexpected changes, homozygous deletion of the floxed exons in DP cells caused a phenotype resembling, albeit weaker than, that resulting from overexpressing the BAF57 dominant-negative mutant lacking the first 133 aa, confirming that Baf57 F is a conditional hypomorphic allele. We are now extending the analysis to other tissues, by deleting the floxed exons from the germline. We suspect that the mice lacking the two exons will be viable, but may display some specific defects. This allele may thus enable us to interrogate the role of BAF57 in a way not feasible with any BAF57 null allele, whether the null is constitutive or conditional. Finally, our ultimate goal of developing this method is to study the potential role of BAF57 in epigenetic programming of mature T cells. Although no gross functional defects in mature T cells were detected in Baf57 F/ΔR ; CD4-Cre; R26 FlpoER1 mice, some specific, subtle defects may exist. Because the mature T cells are resting, it would be possible to test whether restoring BAF57 expression in these cells can rescue the pre-existing phenotype, and we are therefore systematically searching for the putative functional defects in the mature T cells.
Reversible regulation of endogenous genes in mice is necessary for addressing multiple important biological questions. We have combined the Cre/Lox and gene-trap systems to develop LOFT, a reliable and straightforward reversible cKO method. LOFT lacks the limitations of the pre-existing reversible gene regulatory systems, and can also be used to producing traditional constitutive KO and cKO mice. It offers an advantageous alternative to the conventional gene-targeting methods.
DNA construct, embryonic stem cell targeting, and mouse breeding
Primers used in experiments
Sequence (5' → 3')
Screening ES cells
Left arm F
Left arm R
Right arm F
Right arm R
Nested PCR for amplicons produced by RT-PCR
Production of probe for Southern blot
Reverse transcriptase PCR
Total RNA was isolated from sorted CD4 cells with RNAeasy plus (Qiagen) and amplified by a one-step RT-PCR kit (Qiagen Inc., Valencia, CA, USA). The amplicons were re-amplified by nested PCR, gel-purified, and digested with EcoN1.
Southern and western blotting
Genomic DNA (10 ug) was digested with EcoRV and run on a 0.8% agarose gel. The probe was a 1.5 kb PCR product amplified with the primer pair shown in Table 1.
For western blotting, 0.3 million cells were run on a gel (NuPAGE® Novex 4-12% Bis-Tris gel; Invitrogen Corp., Carlsbad, CA, USA) in MOPS running buffer using a commercial protein standard (BenchMark™ Prestained Protein Standard; Invitrogen) as the molecular weight marker. The membrane was probed with a BAF57 antibody directed against the C-terminus of BAF57, before re-probing with an anti-tubulin antibody as loading control. The primary antibodies were detected using horseradish peroxidase-conjugated secondary antibodies, which were visualized with enhanced chemiluminescence reagents on radiography films.
Tamoxifen injection and flow cytometric analysis
TAM solution (20 mg/ml) was prepared by dissolving 200 mg TAM (free base; T5648; Sigma-Aldrich, St Louis, MO, USA) to 0.5 ml ethanol before adding 9.5 ml autoclaved peanut oil. The solution was sonicated and stored at -20°C. To delete the gene-trap cassette, 100 ul of the solution was injected intraperitoneally into adult BAF57 F/ΔR ; Cre; R26 R26FlpoER1 mice once a day for 3 consecutive days. To monitor the effect of TAM, a few drops of peripheral blood were treated with red blood cell lysis buffer, and the cells were then stained with anti-CD4-APC, anti-CD8-PE-Cy7, and anti-B220-PE before flow cytometric analysis of GFP expression in lymphocytes. To determine the effects of BAF57 mutation on early T-cell development, thymocytes were stained with anti-CD4-APC, anti-CD8-PE-Cy7, anti-CD25-PE, anti-CD44-FITC and anti-CD3-Pacific blue, and the data, collected on the flow cytometers (LSRII; BD Biosciences Inc., San Jose, CA, USA), were analyzed as described previously [21, 24, 34].
We thank Drs. Alexandra L. Joyner, Chris Wilson and Klaus Rajewsky for reagents, and João Pereira and Ana Cordeiro Gomes for technical assistance and discussion.
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