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1 Kihara Institute for Biological Research, Graduate School of Integrated Science, Yokohama City University, Maioka-cho 641-12, Totsuka-ku, Yokohama 244-0813, Japan
2 Department of Environment and Natural Sciences, Graduate School of Environment and Information Sciences, Yokohama National University, Tokiwadai 79-5, Hodogaya-ku, Yokohama 240-8501, Japan
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Abstract |
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 Top Abstract IntroductionResultsDiscussionExperimental proceduresReferences |
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Introduction |
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TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
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The gene targeting efficiency is expressed as the ratio of targeted integration frequency to targeted plus random integration frequencies. Therefore, to improve gene targeting efficiency, increasing the former frequency and/or decreasing the latter is needed. Since gene targeting is mediated by cellular HR activity, stimulation of the activity could increase the targeting frequency. HR relies on a number of genes such as RAD51, RAD51 paralogs, RAD52 and RAD54 (Paques & Haber 1999; West 2003). Among these, RAD51 plays a central role in HR reactions: it binds cooperatively to a 3' single-stranded DNA overhang, forms a nucleoprotein filament and promotes homologous pairing through strand invasion and exchange, in collaboration with the additional proteins (Baumann et al. 1996; Arnaudeau et al. 1999). Based on these findings, one strategy for enhancing gene targeting may be to over-express these HR proteins (Vasquez et al. 2001). In fact, over-expression of mouse, hamster and human RAD51 proteins (Vispe et al. 1998; Lambert & Lopez 2000), or human Rad52 protein (Park 1995) has been reported to stimulate intrachromosomal and interchromosomal HR in monkey or hamster cells. However, this stimulation is not for the effects on gene targeting. Yanez & Porter (1999) reported that over-expression of human RAD51 enhances the absolute targeting frequency at the HPRT locus two- to threefold in human HT1080 cells. However, this strategy is not readily applicable to common cell lines, since the cells should be manipulated prior to gene targeting. Another strong candidate for enhancing the frequency would be to inactivate BLM, the gene mutated in Bloom syndrome (BS). BLM is a member of the RecQ family, which is highly conserved from Escherichia coli to man (Ellis et al. 1995; Hickson 2003). BS is a recessive genetic disorder associated with dwarfism, immunodeficiency and cancer disposition (German 1993; Hickson 2003). BLM has been implicated in HR-mediated repair of double-strand breaks occurring at sites of stalled replication forks (Wu & Hickson 2003). BLM is thought to promote the resolution of HR intermediates and to suppress crossovers, thereby blocking abnormal recombination events. Like cells from BS patients, BLM knockout cells from murine ES (Luo et al. 2000), chicken DT40 (Wang et al. 2000), or human HCT116 (Traverso et al. 2003) and Nalm-6 (So et al. 2004) cell lines all display hyperrecombinogenic phenotypes, as evidenced by their increased levels of sister chromatid exchanges (SCEs). Importantly, the knockout cells revealed 3- to 21-fold higher gene targeting efficiencies at various loci tested (Luo et al. 2000; Wang et al. 2000; Traverso et al. 2003; Adachi et al. 2006). In addition, deletion of SGS1, the yeast homolog of BLM, was reported to significantly increase the frequency of targeted gene replacement in yeast (Langston & Symington 2005). We therefore reasoned that transient suppression of BLM expression would enhance HR, leading to an increase in gene targeting efficiency.
In this study, we constructed a gene correction assay system to measure accurately gene targeting efficiencies using the HPRT locus in human HT1080 cells, synthesized small interfering RNA (siRNA) that effectively silence BLM expression and examined whether siRNA transfection could stimulate gene targeting. We show that BLM suppression by siRNA leads to increased gene targeting efficiencies at the HPRT locus. We also show using Nalm-6 cells that BLM siRNA treatment stimulates gene targeting at the HPRT and APRT loci. Therefore, our finding should facilitate a further improvement of protocols to generate knockout mutants from human cell lines.
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Results |
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To measure accurately gene targeting frequencies, we designed a gene correction assay system utilizing the X-chromosome-linked HPRT locus in human HT1080 cells. Figure 1A illustrates the construction scheme of the assay system, which relies on gene correction events by targeted HR at a disrupted HPRT locus. Wild-type (WT) HT1080 cells have a single copy of the HPRT gene, since they are of male origin and proficient in HPRT. To disrupt HPRT, we first transfected WT cells with the pHPRTneo gene disruption vector (for vector construction, see Experimental procedures) by electroporation as previously described (So et al. 2004). The cells were grown overnight, selected with G418 (0.5 mg units/mL) and, 5 days later, subjected to a further selection with 20 µM 6-thioguanine (6TG), a hypoxanthine analog that kills HPRT-proficient cells. The resulting 6TG- and G418-resistant colonies were isolated and screened for targeted cells by Southern blot analysis. Digestion of genomic DNA with NcoI and XhoI gives rise to a 7.2 kb band for WT cells and a 7.5 kb band for correctly targeted cells (Fig. 1A). One of these colonies, designated as C21 (Fig. 1B), exhibited the diagnostic, 7.5 kb band and was unable to survive in HAT medium (HAT-sensitive), confirming HPRT-deficient (HPRT–) targeted cells (disrupted locus I). Subsequently, the neor gene present in C21 cells was removed by transient expression of Cre recombinase by transfecting plasmid pCre as described (So et al. 2004), yielding a cell line designated as d1 (disrupted locus II). The absence of the neor gene in d1 cells was verified by the appearance of a 9.8 kb band in place of the 7.5 kb band (Fig. 1B).
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In gene correction assays, d1 or d1-ß18 cells were transfected by electroporation with the linearized pHPRTpur vector for gene correction, under the same conditions as described above for gene disruption, cultured for 22 h in growth medium, and then selected for colonies that grew in growth medium containing HAT (HAT-resistant; HATr) (Fig. 1A). To confirm that HATr cells resulted from HR events, some of HATr colonies from d1 cells were picked up, grown to mass culture and used to prepare genomic DNA. The structure of the HPRT gene in each clone was examined by PCR analysis using a primer set (3F and 3R) for the sequences flanking exon 3 (Fig. 1A). WT cells displayed a 459-bp band whereas d1 cells a 544-bp disrupted band due to insertion of the loxP sequence (Fig. 1C). In representative correctants 1 and 2, the wild-type 459-bp band was observed in place of the disrupted band seen in d1 cells. Mock-transfected cells (> 107) gave no HATr colonies. Therefore, this assay system permitted us to measure the gene targeting frequency easily, by counting resulting HATr colonies without subjecting to Southern blot or PCR analysis. In addition, the random integration frequency was determined by incubating a portion of transfected cells in puromycin-containing growth medium and counting the resulting puromycin-resistant colonies.
Suppression of BLM expression by siRNA
To search for an effective siRNA that suppresses BLM expression, we synthesized several siRNAs corresponding to distinct regions of BLM mRNA. d1 cells were transfected with each siRNA by lipofection, cultured for 44 h, and assayed for BLM levels by Western blotting using anti-human BLM antibody. We found the greatest reduction of the expression with an siRNA complementary to the BLM mRNA sequence at nucleotide positions 51–69. The duplex siRNA was:
5'-GCUAGGAGUCUGCGUGCGAdTdT-3', 3'-dTdTCGAUCCUCAGACGCACGCU-5'.
Since lipofection was inconvenient and costly for gene targeting experiments, we employed electroporation for transfection and determined an optimal condition for blocking efficiently BLM expression with the effective siRNA. d1 cells were electroporated with 7.5 or 25 µM BLM siRNA, cultured in growth medium for 44 h, and assayed for BLM levels. As shown in Fig. 2A, a marked suppression by BLM siRNA was observed in a dose-dependent manner, i.e. the BLM levels in cells transfected with 7.5 and 25 µM BLM siRNA were reduced to approximately 25% and less than 10% of that in control cells, respectively. Transfection with 25 µM siRNA targeting firefly (Photinus pyralis) luciferase (GL3 siRNA) or no siRNA (mock transfection, MT) did not affect the level of BLM protein in d1 cells. A similar effect was observed in d1-ß18 cells heterozygous for BLM. d1-ß18 cells transfected with 7.5 µM GL3 or no siRNA revealed lower levels of BLM protein than d1 cells treated in the same way (Fig. 2B). Importantly, a great suppression was observed following transfection of d1-ß18 cells with 2.5 µM siRNA, although the suppressed level was similar to that in d1 cells treated with 25 µM. We next examined the time course of the siRNA effect in d1 and d1-ß18 cells electroporated with 25 and 2.5 µM BLM siRNA, respectively (Fig. 2C,D). In both cell lines, the BLM levels were reduced maximally during 22–44 h after transfection, and the levels recovered 20–50% at 66 h. Overall, these results indicate the efficacy of BLM siRNA to suppress BLM expression in the HT1080-derived cell lines under the present experimental conditions.
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Stimulation of gene targeting by BLM siRNA in HT1080 cells
Using the gene correction assay system constructed above, we examined whether transfection of BLM siRNA would stimulate gene targeting. d1 cells were electroporated with 25 µM BLM siRNA, cultured for 45 h in growth medium, and then coelectroporated with the same amount of the siRNA and 4 µg of gene correction vector pHPRTpur. After an additional 22-h incubation, gene corrected colonies and purr colonies were selected in media containing HAT and puromycin, respectively. Table 1 summarizes the results of three independent experiments. The gene targeting frequency in mock-transfected and GL3 siRNA-transfected cells was similar (2.3 x 10–6 and 2.9 x 10–6, respectively), while the frequency in BLM siRNA-transfected cells was elevated to 7.5 x 10–6. In contrast, there was no difference in random integration frequency; i.e. the frequencies of purr clones observed in cells transfected with no siRNA, GL3 siRNA, and BLM siRNA were 1.2–1.4 x 10–3. Therefore, in d1 cells treated with BLM siRNA, the GT/RI ratio (targeting efficiency) was increased threefold from 0.18% to 0.53%. This result indicates that suppression of BLM expression by BLM siRNA enhances the gene targeting efficiency in d1 cells. Interestingly, BLM siRNA at 7.5 µM did not significantly increase the frequency, although the level of BLM protein was considerably decreased (Fig. 2A). The reason for this is not known, but there might be a threshold in the cellular BLM level for leading to an increased targeting frequency, and the concentration of 7.5 µM might be insufficient to suppress the BLM expression over this threshold. Unexpectedly, transfection with the pHPRTpur vector alone following the siRNA pretreatment showed no increase in targeting frequency (unpublished data).
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To further confirm the stimulation of gene targeting by BLM siRNA, we employed another human cell line Nalm-6, which is highly proficient in gene targeting (Grawunder et al. 1998; So et al. 2004; Adachi et al. 2005). For this purpose, we constructed a gene correction assay system using the HPRT locus in Nalm-6 cells, similar to the above-mentioned system for HT1080 cells. We made another disruption vector pHPRThyg, which possesses a hygromycin resistance (hygr) gene and diphtheria toxin A (DT-A) gene as a positive and negative, respectively, selection marker (Supplementary Fig. S2). This vector was used to disrupt the HPRT locus, yielding an HPRT– cell line, W106. W106 cells were transfected with 7.5 µM siRNA alone, cultured for 24 h, and then co-transfected with 7.5 µM siRNA and 4 µg of pHPRTpur (see Experimental procedures). As summarized in Table 3, the targeting frequency was considerably elevated, whereas the random integration frequency was marginally affected. Therefore, the targeting efficiency in BLM siRNA-transfected cells was 6.3-fold higher than that in control cells. Consistent with this stimulation, BLM expression was markedly reduced by BLM siRNA in W106 cells (Supplementary Fig. S3).
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Discussion |
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TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
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To overcome the inefficiency of gene targeting experiments, a variety of strategies have been reviewed extensively (Yanez & Porter 1998; Vasquez et al. 2001). To our knowledge, the present strategy is the first time an siRNA has been utilized to stimulate gene targeting. The strategy is based on the recent findings that BLM knockout cell lines exhibit elevated targeting efficiencies (Luo et al. 2000; Wang et al. 2000; Traverso et al. 2003; Adachi et al. 2006). Our expectation that BLM knockdown by siRNA would exhibit a similar effect has been substantiated. That is, transfection of d1 cells (HPRT–HT1080 cells) with BLM siRNA markedly suppressed BLM expression (Fig. 2A,C) and enhanced the gene targeting frequency threefold (Table 1), whereas the random integration frequency was marginally affected. In BLM+/– d1-ß18 cells, the siRNA effect was more striking: BLM expression was strongly suppressed (Fig. 2B,D) and the targeting frequency was increased 5.1- to 6.0-fold (4.4- to 5.0-fold increase in the targeting efficiency) (Table 2), without influencing the random integration frequency. It should be noted that d1-ß18 cells exhibited an even greater enhancement of targeting efficiency following transfection with one-tenth the amount of BLM siRNA used for d1 cells. Since d1-ß18 cells are heterozygous for BLM, this greater siRNA effect should be attributed to the lowered level of BLM expression compared with BLM+/+ d1 cells (Fig. 2A,B). siRNA acts on cells by specifically degrading mRNA for a gene, but unlikely suppresses its expression completely (Elbashir et al. 2001). Actually, transfection of d1 and d1-ß18 cells with 25 and 2.5 µM BLM siRNA, respectively, appears to leave around 10% of the BLM level seen in the respective mock-transfected cells. Notably, however, the levels of enhancement of gene targeting were comparable to those reported in BLM-null mouse ES (Luo et al. 2000), chicken DT40 (Wang et al. 2000) and human HCT116 (Traverso et al. 2003) cell lines. This may imply that complete loss of BLM expression is not required for enhancing gene targeting efficiencies.
We have also shown using another human cell line, Nalm-6, that suppression of BLM expression leads to a 6.3-fold increase in targeting efficiency in a similar gene correction assay at a disrupted HPRT locus (Table 3) and that the suppression stimulated by over 5.8-fold the targeted disruption of the APRT gene (Table 4).
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Experimental procedures |
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The human fibrosarcoma cell line HT1080 was obtained from Institution for Fermentation (Osaka, Japan). HT1080 cells and their derivatives were maintained at 37 °C in ES medium (Nissui Seiyaku, Tokyo) supplemented with 10% heat-inactivated calf serum (growth medium) in a 5% CO2 incubator. For passage, cells at late-log phase were washed once with Ca2+- and Mg2+-free Phosphate-Buffered Saline (PBS–), and dispersed with 0.1% trypsin/PBS– containing 0.02% EDTA for 5 min at 37 °C; the cells were collected by low-speed centrifugation, resuspended in fresh growth medium at an appropriate density, replated on to 60- or 100-mm tissue culture dishes (NUNC, Roskilde, Denmark), and cultured. The human pre-B cell line Nalm-6 and its derivatives were maintained as described (So et al. 2004).
Vector construction
To construct targeting vector pHPRTneo for disruption of the hypoxanthine phosphoribosyltransferase (HPRT) gene, an 8.9 kb human HPRT gene fragment containing exons 2 and 3 was excised from a BAC clone bWXD187 (Washington University School of Medicine, Department of Molecular Microbiology, Genome Sequencing Center, St. Louis, MO, USA) and subcloned into pBluescript II SK(–). The vector was produced by inserting the neomycin resistance (neor) gene flanked by loxP sequences into the XhoI site present in exon 3 (Fig. 1A). Likewise, pHPRThyg was constructed by inserting the hygr gene into the same XhoI site. Also the DT-A gene cassette, excised from pMC1DT-ApA (Kurabo, Osaka, Japan), was added to the 5'-terminus of the HPRT sequence (Supplementary Fig. S2A). A gene correction vector pHPRTpur was constructed by adding the puromycin resistance (purr) gene to the 3' terminus of the 8.9 kb HPRT fragment (Fig. 1A). An APRT gene disruption vector, pAPRThyg was designed to replace part of exon 3 and entire exon 4 with the hygr gene. For this vector construction, 3.4 kb and 3.3 kb sequences of 5'-arm and 3'-arm, respectively, were amplified by PCR using Nalm-6 genomic DNA as a template (Fig. 3). The primers used were 5'-GCATCTAGATTGGTTGTTGAAGGGCAGGACTGC-3' and 5'-GCAAAGCTTGAATAGGAGGCCCCACAGAGTG-3' for 5'-arm, and 5'-CGCATCGATGGATGCAGCTTACTGTTGTCCAG-3' and 5'-GTCGTCGACCACGGAGAAGCTCACCACTGCTC-3' for 3'-arm. The amplified 3'-arm fragment was subcloned into pMC1DT-ApA, followed by insertion of the hygr gene and the 5'-arm fragment (Fig. 3A). These plasmid vectors were propagated in Eschericia coli HB101 strain and purified with Qiagen Plasmid Maxi Kit (Qiagen K.K., Tokyo).
siRNA transfection
Sense and anti-sense 19-mer RNA oligonucleotides carrying a 3' overhang of dTdT for siRNAs that correspond to different regions of BLM mRNA (GENBANK Accession No. U39817) were synthesized using a DNA/RNA synthesizer (Model 392, Applied Biosystems, Foster City, CA, USA). The oligonucleotides were purified by electrophoresis in a 12% denaturing polyacrylamide gel as previously described (Tanaka et al. 2002) and annealed to duplex siRNAs; the siRNAs were diluted with RNase-free water and stored frozen at –20 °C. As a nonspecific control, an siRNA targeting firefly (Photinus pyralis) luciferase (GL3 siRNA) (Elbashir et al. 2001) was also synthesized. Transfection with siRNAs was carried out by either lipofection or electroporation. Lipofection was performed using Oligofectamine (Invitrogen Japan K.K., Tokyo) according to the protocol of Tuschl (http://www.rockefeller.edu/labheads/tuschl/sirna.html). Electroporation was performed as previously described (So et al. 2004). Briefly, log-phase cells were harvested by trypsinization, washed twice with Saline G (Puck et al. 1958), and electroporated with siRNA alone (or along with targeting vector) in a 40-µL chamber of Electro Gene Transfer Equipment (GTE-1; Shimadzu, Kyoto) containing 4 x 106 cells suspended in fresh Saline G. After 15 min, the cells were plated into 5–8 100-mm dishes each containing 10 mL of growth medium.
Southern blot and PCR analyses
Isolation of genomic DNA from individual clonal cell lines, Southern blot and PCR analyses were carried out as previously described (Sado et al. 2001). For Southern blotting, 10 µg of genomic DNA was digested with an appropriate restriction enzyme, electrophoresed in a 0.8% agarose gel and blotted to an Hybond-N+ membrane (Amersham Biosciences, Piscataway, NJ, USA); hybridization of the membrane was done with probes depicted in Figures. PCR analysis of a corrected HPRT locus was performed using primers 3F (5'-GTGGAAGTTTAATGACTAAGAG-3') and 3R (5'-GTATATATCCTCCAAGGTGACTAG-3'), which are located upstream and downstream, respectively, of exon 3. PCR products were electrophoresed in a 2% agarose gel.
Western blot analysis
Cells transfected with siRNA were rinsed twice with PBS– and scraped in 50–100 µL of lysis buffer [50 mM Tris-HCl (pH 6.8), 2% sodium dodecylsulfate, 10% glycerol, 100 µM dithiothreitol, 1 mM phenylmethylsulfonylfluoride] containing Protease Inhibitor Cocktail (1/10 volume of buffer; Sigma-Aldrich, St. Louis, MO, USA). The lysates were allowed to stand for 20 min at 4 °C and, after sonication, centrifuged for 20 min at 16 000 g. The supernatants were collected and used for Western blot analysis; their protein concentrations were determined by using Bio-Rad Protein Assay (Bio-Rad Laboratories, Hercules, CA, USA) according to the supplier's instructions. Twenty micrograms of the lysates were electrophoresed in a 5 or 10% polyacrylamide gel and then transferred on to a polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA, USA). The membrane was probed with antibodies against human BLM (ab476, Abcam, Cambridge, UK) and actin (Sigma-Aldrich). Proteins were visualized using an ECL Western Blotting Detection System (Amersham Biosciences), and quantified using NIH image software.
Assays of gene targeting and random integration
In HT1080 cells, assays for gene targeting and random integration were carried out using the gene correction assay system shown in Fig. 1. Log-phase cells were harvested by trypsinization, washed twice with Saline G, and resuspended in fresh Saline G; 4 x 106 cells were transfected by electroporation with either BLM siRNA or control GL3 siRNA, or no siRNA, replated on to 4–6 100-mm dishes each containing 10 mL of growth medium and cultured for 45 h. Then, the cells were harvested as above; 4 x 106 cells were again electroporated with the same amount of the siRNA and 4 µg of gene correction vector pHPRTpur linearized with NgoMIV (New England Biolabs, Ipswich, MA, USA). The cells were resuspended in growth medium and counted, and aliquots of the cell suspension were replated on to 3–5 100-mm dishes either at 5–10 x 105 cells/dish to determine gene correction frequency, or at 5–10 x 104 cells/dish to determine random integration frequency. In addition, for determination of plating efficiencies, aliquots of the resting cell suspension were diluted and replated in triplicate at a density of 200 cells/100-mm dish. After a 22-h incubation, to the dishes for the gene correction assay was added HAT (100 µM hypoxanthine, 0.25 µM amethopterin, and 10 µM thymidine), while puromycin 2HCl (1 µg/mL, Wako Pure Chemical, Osaka, Japan) was added to the dishes for the random integration assay. After a 10- to 12-day cultivation, the resulting colonies were fixed with 10% formaldehyde in saline, stained with 0.1% crystal violet, and counted; at the same time, some of the colonies grown in HAT medium were isolated by trypsinization in a steel cylinder and expanded for structure analysis. Gene targeting and random integration frequencies were calculated as the number of HAT-resistant and purr colonies, respectively, divided by the number of plated cells multiplied by their plating efficiencies.
In Nalm-6 cells, gene correction assays were carried out in essentially the same manner as in HT1080 cells. Briefly, one of the HPRT– clones (W106) generated by gene disruption with pHPRThyg (see Supplementary Fig. S2B) was transfected by electroporation with 7.5 µM BLM siRNA or GL3 siRNA and cultured in fresh growth medium. Twenty-four hours later, the cells were co-transfected with the same amount of the siRNA and 4 µg of pHPRT-pur. After an additional 24-h incubation, the cells were replated at a density of 5–10 x 105 per 100-mm bacterial dish into agarose medium containing either HAT or 0.5 µg/mL puromycin. After a 2-week incubation, the resulting colonies were counted.
For targeted gene disruption of the APRT locus, Nalm-6 cells were electoroporated with 7.5 µM BLM siRNA or no siRNA (MT) and cultured. After a 24-h incubation, the cells were harvested and co-transfected with the same amount of the siRNA and 4 µg of pAPRThyg. After an additional 24-h incubation, the cells were replated at a density of 5–10 x 105 per 100-mm dish into agarose medium containing 0.4 mg/mL hygromycin B (Wako Pure Chemical). After a 2-week incubation, the resulting colonies were isolated and expanded to prepare genomic DNA. Gene targeting events were confirmed by Southern blot analysis.
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Acknowledgements |
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Footnotes |
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These authors contributed equally to this work. 
* Correspondence: E-mail: koyama{at}yokohama-cu.ac.jp |
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Received: 1 September 2005
Accepted: 21 December 2005
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