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[10] Targeting Cellular Genes with PCR Cassettes Expressing Short Interfering RNAs By Daniela Castanotto and Lisa Scherer Abstract

The synthesis and transfection of PCR short interfering/short hairpin RNA (si/shRNA) expression cassettes described in this chapter can be used to rapidly test siRNA targeting and function in cells. One critical element in the design of effective siRNAs is the selection of siRNA–target sequence combinations that yield the best inhibitory activity. This can be accomplished by using synthetic siRNAs and transfection procedures, but these can be costly and time consuming. By using the PCR strategy, it is possible to create several expression cassettes and simultaneously screen for the best target sites on any given mRNA. This PCR strategy allows a rapid and inexpensive approach for intracellular expression of si/shRNAs and subsequent testing of target site sensitivity to downregulation by RNA interference (RNAi). Introduction

At present, RNA interference (RNAi) is perhaps the most powerful genetic tool for target-specific knockdown in mammalian cell (Hannon, 2002; Tuschl, 2001). siRNAs and the related short hairpin RNAs (shRNAs)

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can be expressed in vivo against human, viral, and cellular targets. The most popular method for targeting a gene is based on transfection of chemically synthesized siRNA duplexes or intracellular expression of siRNAs using RNA polymerase III and polymerase II (pol III and pol II) cassettes (Elbashir et al., 2001a,b; Tuschl, 2001, 2002). Despite the great potential of siRNAs, identifying optimal siRNAs target sites can be problematic, which is critical for siRNAs function (Holen et al., 2002; Lee et al., 2002) and specificity. Although these molecules are generally highly specific, in high concentration they can elicit nonspecific off-target effects. Therefore, it is critical to identify the best target site – siRNA combination that provides the highest knockdown at the lowest possible concentration of siRNAs. Moreover, at high concentrations, it is possible that some siRNAs can activate the interferon response pathway, which could lead to nonspecific degradation of cellular RNA (Bridge et al., 2003; Sledz et al., 2003). Several Web sites contain programs that use algorithms to find the best siRNA design for a specific target. Combining these programs with new rules for the thermodynamic properties of the sense and antisense siRNAs (Khvorova et al., 2003; Schwarz et al., 2003) is helpful for designing effective interfering/short hairpin RNAs (si/shRNAs), but the best target site selection can only be found by systematically and empirically testing a variety of potential targets (Vickers et al., 2003). However, constructing several vectors expressing si/shRNAs to test their downregulation activity in vivo is considerably time consuming. One possible way of overcoming this limitation is to have a simple screening procedure that allows the testing of several different sites along a messenger RNA (mRNA) for sensitivity to siRNA. The PCR-based method previously described (Castanotto et al., 2002) and detailed here has proven to be both straightforward and effective. The method involves creation of pol III transcription units by PCR and direct transfection and testing of the PCR products for siRNA function in cell culture. Design of Small Interfering RNA (siRNA)-Expressing PCR Cassettes

The expressed siRNA may consist of two separate, annealed single strands of 21 nt, in which the terminal two 30 nucleotides are unpaired (30 overhang) or it may be in the form of a single stem-loop, often referred to as a shRNA. siRNAs exogenously introduced into mammalian cells usually, but not always (Sledz et al., 2003), bypass the Dicer step and directly activate homologous mRNA degradation without initiating the interferon response (Caplen et al., 2002; Elbashir et al., 2001a; Harborth et al., 2001), whereas double-stranded RNA (dsRNA) longer than 30 nt triggers the nonspecific interferon pathway. Activation of the interferon pathway can

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lead to global downregulation of translation as well as global RNA degradation. Thus, it is advisable to design the siRNA sense and antisense strands to be shorter than 30 nt. Duplexes of 21 nt are usually sufficient to obtain full activity of expressed si/shRNAs. However, slightly longer (24–27) si/ shRNAs, which undergo processing to 21-nt molecules by the Dicer enzyme, could be more efficiently incorporated into the RNA-induced silencing complex (RISC) and thus more effective in downregulating their target. With the exception of the two nucleotides that form the 30 overhang, the antisense sequence should be completely complementary to the target to direct its cleavage. However, some mismatches can be tolerated [Amarzguioui et al., 2003; Bernstein et al., 2001; Brummelkamp et al., 2002; Chiu and Rana, 2002; Dohjima et al., 2003; Elbashir et al., 2001; Holen et al., 2002; Randall and Rice, 2001; Yu et al., 2002; Hutvagner and Zamore, 2002] and this should be considered when designing inactive, mutant forms of the siRNAs for use as negative controls. In general, we find that four consecutive nucleotide mismatches in the center of the antisense strand work for the majority of siRNA–target combinations. Standard computer searching programs (e.g., BLAST) should also be used to exclude the presence of long stretches of siRNA complementarity with nontargeted cellular genes. Because U6 initiates transcription with a G, it will be incorporated as the first base of the sense or antisense strand. For this reason, in the design of siRNAs we prefer placing the sense strand preceding the antisense strand, because the active strand (the antisense) will not obligatorily contain a 50 G. If an extra G is included at the 50 end of the sense strand, it is not necessary to include a corresponding C at the 30 end of the antisense strand because the G can form a G–U pair with one of the 30 terminal Us that serve as a termination signal for RNA pol III; it is also important that the si/shRNAs does not include more than three Us in a row. Thus, the selected target site (and the sense strand) should not contain more then three contiguous As. Construction of si/shRNA PCR Cassettes

PCR Oligo Design The procedure for the PCR approach employs a universal primer complementary to the 50 end of the U6 promoter (other pol III and possibly pol II promoters can also be used) along with a unique primer complementary to the 30 end of the promoter. The 30 primers can be designed to include complementary sequences to either the sense or antisense siRNAs or to include both the sense and antisense sequences linked by a short loop. The first design is used when two separate PCR cassettes independently expressing the sense and antisense siRNA are constructed (Fig. 1A). The sense or

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Fig. 1. Schematic representation of the PCR strategy used to generate U6 transcription cassettes expressing siRNAs. The 50 PCR primer is complementary to the 50 end of the U6 promoter and is standard for all PCR reactions. (A) The 30 PCR primer is complementary to sequences at the 30 end of the U6 promoter followed by either the sense or antisense sequences, a stretch of six deoxyadenosines (Ter), and an additional stuffer-tag sequence, which contains a restriction site and six additional random nucleotides. The As are complementary to the termination signal for the U6 pol III promoter; therefore, any sequence added after this signal will not be transcribed by the pol III polymerase and will not be part of the siRNA. (B) The first 30 PCR primer is complementary to sequences at the 30 end of the U6 promoter followed by the sense sequences and a 9-nt loop. The second 30 PCR primer contains a sequence complementary to the 9-nt loop followed by the antisense sequences, a stretch of four to six deoxyadenosines (Ter), and the additional stuffer-tag sequence. The sense and antisense sequences are inserted in the cassette by a two-step PCR reaction (see text). (C) The sense and antisense sequences, linked by a 9-nt loop and followed by the stretch

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the antisense sequences are followed by a stretch of six deoxyadenosines (Ter) and by a short additional sequence, which we term the stuffer-tag sequence, which includes a restriction site for possible cloning at a later stage as well as six random nucleotides. Thus, the resulting PCR products include the U6 promoter sequence, the sense or antisense, a terminator sequence, and the selected restriction sites at the 50 and 30 termini of the product. The two PCR cassettes making the sense or the antisense siRNA strands should be simultaneously cotransfected in cells to test their activity. The best combination among the sets of cassettes tested can be later cloned into a single expression vector for long-term studies. To maximize intracellular expression and avoid promoter interference, it is advisable to clone the two U6 cassettes expressing the sense or antisense siRNA strand in the same orientation and separated by a short spacer. If the selected vector backbone does not contain suitable restriction sites, it is possible to include the spacer in the design of the 50 PCR oligonucleotide by starting with a restriction site at the 50 end of the oligo and proceeding with the spacer and with the nucleotides complementary to U6 promoter sequences. A preferable design is to include both the sense and antisense sequences within the same cassette (Fig. 1B, C). In this approach, the 30 primer contains sequences that encode the complete shRNA, a pol III terminator sequence (T6), a restriction site for possible cloning at a later stage, as well as four to six random nucleotides. The sense and antisense siRNA sequences contained in the 30 primer are linked with a short 9-nt loop (UUUGUGUAG), which in our experience has proven to be an effective loop with several different targets. However, a number of other loop sequences are suitable for siRNA function. When selecting a loop sequence it is advisable to base the selection on comparison among various microRNA sequences, because naturally evolved sequences are likely to be processed more efficiently. If the UUUGUGUAG loop sequence is selected for the shRNA design, it is important that the siRNA sense strand does not contain a U at its 30 terminus as this would create a stretch of four Us that could serve as a pol III terminator element. To construct the U6-shRNA cassette, two 30 primers or a single 30 primer can be used. When two 30 primers are used, the first PCR reaction employs the 50 U6 universal primer and a 30 primer complementary to 25 nt of the U6 promoter, followed by sequences complementary to the sense and the 9-nt loop (Fig. 1B). One microliter of this first reaction is reamplified in a second PCR reaction that employs the same 50 U6 primer and of As and the stuffer-tag sequences, are included in a single 30 primer. (D) Complete PCR expression cassette obtained by the PCR reaction. To increase the yield of the PCR product shown in (B), an additional PCR step can be performed using the universal 50 U6 primer and a 30 primer complementary to the Tag sequence, as indicated in the figure and in the text.

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a 30 primer harboring sequences complementary to the 9-nt loop appended to the antisense strand, Ter, and restriction site sequence (stuffer-tag sequence, Fig. 1B). It is possible to add a few additional nucleotides complementary to the sense strand after the 9-nt loop to increase the Tm of this second PCR primer; however, it is important to use caution in designing the extended complementarities because this will cause snapback at the 30 end of the PCR primer and create difficulties in the extension step. The resulting PCR products include the U6 promoter, the sense and antisense coding sequences, followed by the pol III terminator sequence and the restriction site (Fig. 1D). When a single 30 primer is used, the procedure consists of a one-step PCR reaction with a 30 primer containing sequences complementary to the 30 end of the promoter and sequences complementary to the sense, the loop, and the antisense siRNA gene followed by a stretch of six deoxyadenosines (Ter) and by the short sequence that includes the restriction site and six random nucleotides (Fig. 1C). The resulting PCR product includes the U6 promoter, the sense and antisense siRNAs in the form of a stem loop, the terminator sequence, and the restriction sites (Fig. 1D). This second approach employs a considerably long and structured 30 PCR primer that with a few sequences may cause difficulties in the amplification reaction, in which case it is helpful to slightly modify the standard amplification conditions (see next section). However, this strategy minimizes the possibility of inserting any polymerase-induced mutations in the siRNA sequence during the amplification reaction. In addition, the RNA expressed intracellularly by this PCR cassette is already in the form of a stem loop, which should have greater stability than when the sense and antisense siRNAs are expressed from two separate cassettes as single-stranded RNAs. A number of commercial DNA synthesis facilities are capable of synthesizing the resulting 30 primers, which are typically 85–95 nt. If required, there are several options for primer purification. HPLC purification is efficient, but not recommended because it has poor resolution for primers of this length. Urea-PAGE purification has higher resolution, but yields are often low and if not done carefully can introduce contaminants that interfere with subsequent PCR reactions. However, it is a procedure that can be easily performed in most laboratories. In practice, however, simple desalting should be sufficient. Generally, most failure sequences will reflect incomplete products from the last rounds of synthesis of the 50 end of the primer, which encodes the 30 end of the PCR product. Designing the primer with sufficient 30 noncoding sequence, along with purification of the final PCR product (see later), should minimize this potential problem. For direct transfection and testing of the PCR-amplified siRNA genes, the 50 termini of the PCR primers must be phosphorylated by using DNA

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polynucleotide kinase and nonradioactive ATP. This modification results in enhanced function of the PCR products, perhaps by stabilizing them intracellularly or promoting enzymatic ligation and multimerization of the PCR cassettes. PCR primers are phosphorylated before the PCR reactions, using a 600-pmole primer, 1 buffer (New England Biolabs, Beverly, MA), 1 mM ATP, and 20 units T4 polynucleotide kinase in a total volume of 100 l. After a 30-min incubation at 37, reactions are adjusted to 2.0 M ammonium acetate, extracted once with phenol:chloroform:isoamyl alcohol (25:24:1), once with chloroform:isoamyl alcohol (24:1), and precipitated with 2.5 volumes of ethanol and glycogen carrier (Roche, Switzerland). After collecting precipitates by centrifugation, the resulting pellets are washed in 70% ethanol, dried briefly, and resuspended at 50 pmoles/ml. Alternatively, some companies that synthesize oligos provide the option of phosphorylating them as well, which may save both time and money when it is anticipated that an oligo (such as the universal 50 oligo) is to be used repeatedly. PCR Reaction Conditions PCR reactions use a hot-start protocol, beginning with a mix of 1 Vent polymerase buffer (New England Biolabs), 40 nmoles of each dNTP, and 100 pmoles of each of the universal U6 50 and unique 30 primers in a final volume of 25 l in a PCR tube containing an Ampliwax pellet, which is then incubated at 85 for 10 min, followed by a brief cooling to harden the wax layer. The top mix consists of 75 l of 1 Vent buffer, 2 units Vent polymerase, and 1 g of a template plasmid containing the desired promoter, preferably linearized immediately downstream of the transcription start site. Although linearizing of the template in this manner is not required, it can increase the efficiency of amplification. In most cases, a simple PCR reaction that cycles 1 min at 95, 1 min at 55, and 1 min at 72 is sufficient to obtain enough product for functional tests in mammalian cells. When specific sequences present difficulties in the amplification reaction, it is preferable to perform a ramp PCR, in which the cycling parameters are typically 1 min at 95, 3–5 min slow cool (ramping down) to the annealing temperature, 1 min at the final annealing temperature followed by 3–4 min ramping up to the extension temperature of 82, and 1 min at 82. The final annealing temperature is determined by the 30 primer and should be at least 10 below the theoretical Tm for the 30 primer and the U6þ1 template; 15 below the theoretical Tm can increase yield. These reactions give very low yield under typical PCR conditions, in which the annealing temperatures are usually 5 below the theoretical Tm for the primer–template pair with the lowest annealing temperature. We postulate that the 30 primer snaps back into the hairpin conformation very efficiently at a relatively high temperature,

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which may interfere with annealing to the template. For this reason, we recommend at least 22–24 nt of complementarity with the template promoter sequence. (As an aside, snap-back formation may have the favorable effect of reducing the formation of primer dimers.) The occasional 30 primer may have a higher, narrow-range optimum annealing temperature, but, in general, ramping down between the denaturation and annealing steps allows efficient annealing of all primers without time-consuming optimization. The synthesis step is carried out at 82, 10 higher than is usual for Vent polymerase, to allow for more efficient read-through of the stem loop of the 30 primer. Vent has close to maximum activity at this temperature, whereas the enzyme half-life is not significantly affected (personal communication, technical services, New England Biolabs). The ramp-up between the final annealing and synthesis steps is to allow for some extension of the primers, stabilizing interaction with the template and preventing premature dissociation from the template. These reactions are often not as efficient as standard PCR reactions and may require more cycles than theoretically necessary; typically, we use 30 cycles of amplification. For all PCR reactions we primarily use Vent polymerase, because of its high fidelity relative to Taq polymerase. PfuTurbo (Stratagene, La Jolla, CA) can sometimes improve PCR of highly structured templates, which may be useful for difficult shRNA templates. The temperature of the synthesis step with PfuTurbo can also be increased to the 74–78 range. Because PfuTurbo has very little activity between 40 and 50 , the hot-start protocol and slow ramp-up between the annealing and synthesis temperatures is probably unnecessary. Also, this enzyme tolerates addition of DNA denaturants that can improve results (see Stratagene protocols for details); we have used 1% high-quality DMSO with success. However, in the few cases we found difficulties in amplifying the template with Vent or PfuTurboÕ polymerases, the Taq polymerase was effective in yielding sufficient amounts of shRNA PCR cassettes, which were subsequently confirmed to be devoid of mutations. When using Taq polymerase, we have performed synthesis at temperatures up to 75. Finally, a 30 primer designed to be complementary to the short sequence located at the 30 end of the PCR cassette (stuffer-tag sequence) can be used together with the U6 universal primer to increase the production of poorly amplified PCR cassettes (Fig. 1D). The 30 primer includes the pol III terminator (six Ts), the restriction site, and the six additional random nucleotides that follow the restriction site, for a total of 18 nt (Castanotto et al., 2002). This reamplification step should only be performed if all other strategies to improve the yield of the PCR product have failed. When the PCR products appear homogeneous, they can be purified directly using QiaquickÕ PCR columns. These column-purified products

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can be transfected into cells after assessing their concentration with a spectrophotometer. It is not unusual to see an additional band migrating just below the main product band. The proportion of this secondary product relative to the main product is typical of a given construct. The nature of this band is unknown; possibilities include alternative conformers that migrate differently (e.g., a cruciform structure of the shRNA region) or incomplete PCR products. Careful isolation of the upper band does not result in reappearance of the lower band on reanalysis. PCR products are isolated from a 1.6% agarose gel and full-length bands purified by Qiaquick columns (Qiagen), employing the extra washes recommended for microinjection. Recovery is quantified by gel electrophoresis and ethidium bromide staining relative to an internal standard. Transfection Conditions

Transfection conditions depend on the cell system being used. Once the PCR reaction is completed and the products are column purified, they can be applied to cells by using cationic liposomes, calcium phosphate, or electroporation, depending on the cells in question. PCR cassettes become localized to the nucleus, where they will transcribe the shRNA or siRNA molecules capable of downregulating their cellular target (Fig. 2). The purified pol III-PCR cassettes can be either cotransfected with a plasmid expressing a specific target or directed against an endogenous cellular target. With 25–100 ng of the PCR cassette expressing the si/shRNA, 250 ng of the target plasmid can be cotransfected. As little as 25 ng of the PCR product can be effective in producing siRNAs. However, it is advisable to transfect at least 50 ng of PCR product. If co-transfections of the target gene on a plasmid and the PCR products are to be utilized for experiments, the ratio between the PCR products and the plasmid expressing the target should be calculated to fall into molar ratio ranging from 1:1 to 1:5. To facilitate transfection of small amounts of PCRamplified DNA, 400 ng to 1 g of a plasmid such as BlueScriptÕ (Stratagene) should be added to each reaction to serve as carrier. We have had poor success using chromosomal DNAs as carrier. It is important that the total amount of DNA be the same for each transfection. The BlueScript plasmid can be used in each case to achieve the desired amount of DNA recommended for each transfection procedure. For readily transfectable cell lines, transfections can be performed in six-well plates, using Lipofectamine Plus‘ (Life Technologies, GibcoBRL, Gaithersburg, MD) or other transfection reagents as described by the manufacturer. Transfection reagents specifically developed for transfection of PCR cassettes in cell cultures are commercially available (e.g., siPortX-1, Ambion, Austin, TX).

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Fig. 2. Schematic representation of the steps required to inhibit cellular gene expression with pol III-driven shRNA PCR cassettes. A template containing the U6 pol III promoter is amplified with a 30 primer containing the shRNA sequence as described in the text. Within a couple of hours the PCR reaction generates a transcription unit capable of producing functional siRNA. The purified cassette is directly transfected into selected cell lines to target an expressed cellular gene. If the experimental cell line does not produce the shRNA target, the PCR cassettes can be cotransfected with a plasmid expressing the target of choice (see text). The RNAs transcribed from the PCR cassettes fold into the double-stranded shRNA structure, are transported to the cytoplasm where they are processed into siRNAs, and enter RISC, resulting in degradation of their mRNA target. The various siRNAs can be screened for function 24–48 h after transfection.

Strong and specific downregulation of the target gene by the siRNAs should be detected 36–48 h post-transfection. One example of this PCR methodology is shown in Fig. 3. In this experiment, a PCR cassette was used to express a shRNA directed against the HIV-1 rev target gene, which is fused to a gene encoding for the green fluorescence protein. The gene fusion, which can be potentially created for any targeted gene, allows a quick screen for the efficacy of the si/shRNA through monitoring inhibition of fluorescence. A plasmid expressing the red fluorescence protein is cotransfected with the target and the PCR cassettes as a control for transfection efficiency. A control for transfection efficiency should always be included in all transfection experiments, especially if different si/shRNA

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Fig. 3. (A) Downregulation of a rev:egfp gene fusion by an anti-rev shRNA-PCR cassette in 293 cells. The right panels show the cells under a bright field. The middle panel shows the red fluorescence generated by the ds2Red protein-expressing plasmid that was cotransfected with the experimental samples and used to determine the efficiency of transfection for each construct. The left panels show the green fluorescence generated by the intracellular expression of the rev:egfp fusion gene. (Top) The rev:egfp target was cotransfected with the ds2Red plasmid. (Middle) The rev:egfp target was cotransfected with the ds2Red plasmid and with 50 ng of a functional anti-rev shRNA PCR cassette. (Bottom) The rev:egfp plasmid was cotransfected with the ds2Red plasmid and with 50 ng of a shRNA PCR cassette with an irrelevant (nontargeted) sequence. (B) Inhibition of HIV-1 p24 antigen production by the anti-rev shRNA PCR cassette. A total of 100 ng of the anti-rev shRNA cassette was cotransfected with pNL4-3 HIV proviral DNA. A nonfunctional anti-rev shRNA-PCR cassette (siRNA mutant) containing four mutations in the middle of the shRNA sequence was

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cassettes are tested to assess their relative ability to downregulate the target. FACS analyses can be used to obtain the exact levels of green fluorescence protein, which can be normalized to the efficiency of transfection of the various PCR cassettes by using the level of red fluorescence protein detected in each experimental sample (also obtained by FACS). The anti-rev shRNA that proved to be effective in cell cultures against the HIV-1 rev:egfp fusion target (Fig. 3A) was equally effective when used in bona fide HIV-challenge assays (Fig. 3B) and after it was cloned in an expression vector (data not shown). The anti-HIV rev PCR cassette used in cotransfection assays with HIV-1 proviral DNA was able to yield 1000-fold inhibition of p24 antigen production (Fig. 3B). For these assays, we typically use 0.5 g proviral DNA, 100–200 ng of U6 þ 1 shRNA PCR product, and enough pBluescript to bring the total amount of DNA up to 1 g and transfect by using Lipofectamine Plus (Invitrogen, Carlsbad, CA) in OptiMEM, according to the manufacturer’s instructions. With a good target, as little as 25 ng of PCR product can inhibit HIV-1 replication, measured by p24 antigen production. Conclusions

The direct transfection of PCR cassettes allows a simple and rapid screening of many genes and many sites within the same target gene. Using the si/shRNA PCR approach and a 96-well plate for cells transfection, it is potentially feasible to simultaneously test for the accessibility and siRNA sensitivity of 96 sites present in the target gene in a single transfection experiment within a short time frame (Fig. 2), thereby saving time and effort. By using the PCR cassettes, we have observed target inhibition for at least six days. A nonfunctional mutant siRNA or a target with silent codon changes in the region of the siRNA base pairing should always be used as a control for nonspecific effects. Once the PCR product that works best for a given target is identified, it can be easily cloned into a plasmid or viral vector for transfection and transduction into primary cells and to conduct long-term studies of gene knockdown. Acknowledgments This work was supported by NIH grants A129329, AI 42552, and HL074704.

used as control for nonspecific effects. The pTZ U6 þ 1 empty vector (plasmid) was used as a control to exclude nonspecific effects caused by the U6 promoter. The anti-rev shRNA PCR cassette yielded a 1000-fold inhibition of p24 antigen production and inhibited p24 antigen production by HIV-1 for up to 6 days post-transfection.

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