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Detection of Sleeping Beauty transposition in the genome of host cells by non-radioactive Southern blot analysis
Accepted Manuscript Detection of Sleeping Beauty transposition in the genome of host cells by nonradioactive Southern blot analysis Rajagopal N. Aravalli, Chang W. Park, Clifford J. Steer PII:
S0006-291X(16)31012-9
DOI:
10.1016/j.bbrc.2016.06.094
Reference:
YBBRC 36010
To appear in:
Biochemical and Biophysical Research Communications
Received Date: 3 June 2016 Accepted Date: 18 June 2016
Please cite this article as: R.N. Aravalli, C.W. Park, C.J. Steer, Detection of Sleeping Beauty transposition in the genome of host cells by non-radioactive Southern blot analysis, Biochemical and Biophysical Research Communications (2016), doi: 10.1016/j.bbrc.2016.06.094. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Detection of Sleeping Beauty transposition in the genome of host cells by non-radioactive Southern blot analysis
a
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Rajagopal N. Aravalli a,b,1, *,¶, Chang W. Park b,1, and Clifford J. Steer b,c,*
Department of Radiology, University of Minnesota Medical School, MMC 292, 420
b
Department of Medicine, University of Minnesota Medical School, MMC 36, 420 Delaware
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Street SE, Minneapolis, MN 55455, USA c
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Delaware Street SE, Minneapolis, MN 55455, USA
Department of Genetics, Cell Biology and Development, University of Minnesota,
Minneapolis, MN 55455, USA
These authors contributed equally to the work.
*
Corresponding authors
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¶
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E-mail: [email protected]; [email protected]
Present address: Department of Electrical and Computer Engineering, University of
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Minnesota, 200 Union Street SE, Minneapolis, MN 55455, USA
Number of pages: 28 (including the figures) Number of figures: 4
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Abstract The Sleeping Beauty transposon (SB-Tn) system is being used widely as a DNA vector for the delivery of therapeutic transgenes, as well as a tool for the insertional mutagenesis in animal
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models. In order to accurately assess the insertional potential and properties related to the integration of SB it is essential to determine the copy number of SB-Tn in the host genome. Recently developed SB100X transposase has demonstrated an integration rate that was much
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higher than the original SB10 and that of other versions of hyperactive SB transposases, such as HSB3 or HSB17. In this study, we have constructed a series of SB vectors carrying either a
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DsRed or a human β-globin transgene that was encompassed by cHS4 insulator elements, and containing the SB100X transposase gene outside the SB-Tn unit within the same vector in cis configuration. These SB-Tn constructs were introduced into the K-562 erythroid cell line, and their presence in the genomes of host cells was analyzed by Southern blot analysis using nonradioactive probes. Many copies of SB-Tn insertions were detected in host cells regardless of
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transgene sequences or the presence of cHS4 insulator elements. Interestingly, the size difference of 2.4 kb between insulated SB and non-insulated controls did not reflect the proportional difference in copy numbers of inserted SB-Tns. We then attempted methylation-
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sensitive Southern blots to assess the potential influence of cHS4 insulator elements on the
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epigenetic modification of SB-Tn. Our results indicated that SB100X was able to integrate at multiple sites with the number of SB-Tn copies larger than 6 kb in size. In addition, the nonradioactive Southern blot protocols developed here will be useful to detect integrated SB-Tn copies in any mammalian cell type.
Keywords: Epigenetic; Methylation; Sleeping Beauty; SB100X; Southern blot; Transposon
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Introduction Sleeping Beauty (SB) is a member of the Tc1/Mariner superfamily of DNA transposons
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that are widespread in nature, but are transpositionally inactive in vertebrates due primarily to the accumulation of mutations [1]. SB is a synthetic transposon, which was originally constructed based on sequences of transpositionally inactive elements found in fish genomes
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[2]. The resurrected SB was engineered by removing the inactivating mutations from the
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transposase open reading frame. By using high throughput genetic screening of SB variants, a hyperactive version SB100X, that displayed ~ 100-fold higher activity than the originally resurrected transposase, was identified [3]. This SB transposon (SB-Tn) system yielded robust gene transfer efficiencies in a variety of mammalian cells, and enabled highly efficient
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germline transgenesis in relevant animal models of human diseases (reviewed in [1]). We previously reported DNA methylation in both IHK promoter and DsRed transgene
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sequences, which was correlated with the level of IHK-DsRed transgene expression in the
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host genome of K-562 cells after integration via SB-Tn [4] . DNA methylation of transgenes inserted into the host genome has been reported in other studies using retroviral vectors [5,6], which led to progressive DNA methylation and transgene silencing. CpG methylation was also reported in transgenes delivered and integrated into animal cells by SB-Tn systems [4,7,8]. Studies that were able to correlate the expression of transgenes delivered via the SBTn system with the level of DNA methylation demonstrated a predicted inverse relationship
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with expression [4,7]. In an attempt to prevent this epigenetic silencing of the transgenes by DNA methylation, we introduced heterologous chicken HS4 β-globin insulator elements
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(cHS4) into our SB transgene cassettes. This HS4 insulator element was able to block enhancer activity [9,10], thereby either eliminating or diminishing the influence of enhancer elements on genes in close proximity, whether the enhancer was included in the exogenous
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transgene constructs or placed close to the inserted exogenous transgene in the host genome.
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In addition, HS4 insulator is known to protect transgenes from silencing by epigenetic modification, such as CpG methylation when the transgene is shielded by cHS4 insulator elements [11,12]. In a recent report, it was shown that successful expression of the fluorescent transgene can be maintained for long term when the transgene was flanked by the
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cHS4 insulator element in a SB-Tn vector [13]. Furthermore, we could expect the enhancer blocking activity of the insulator to prohibit the potential transactivation of neighboring host
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genes by SB-mediated transgene insertion, which we observed in a few insertion loci [4,14].
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Therefore, we cloned cHS4 insulator elements into our SB vector constructs so that they flank both sides of the transgene cassette within SB transposons defined by IR/DR sequences. Control vectors, which lack just the insulator element, were also generated and tested in comparison with insulated SB constructs. To achieve maximum efficiency of transposon insertion into the host genome, we used SB100X [3]. For all our SB-Tn vectors, whether in cis- or trans-configuration, SB100X was
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the source of transposase, and the expression of the transgenes was initially tested in an erythroid cell line, K-562. Using non-radioactive probes in Southern blotting, we successfully
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detected SB transposons in host cells. As expected from the hyperactive SB transposase SB100X, a large number of insertions of SB transgene cassettes were detected, of which some were greater than 6 kb in size. Additionally, we also confirmed a possibility of assessing
2. Materials and methods
Sleeping Beauty transposon constructs
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2.1
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sensitive non-radioactive Southern blot analyses.
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DNA methylation status of SB constructs containing cHS4 elements by using methylation
The cis pKT2-meIF-SB100X and trans pKT2-RV SB-Tn vectors [15] contained the
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hyperactive SB100X transposase gene driven by the mouse eukaryotic initiation factor 4A1 promoter (InVivogen, San Diego, CA). We first made SB constructs in which SB transposon
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units had two insulator elements (cHS4; [10]) flanking multiple restriction endonuclease recognition sites (MCS) derived from pIRES2-EGFP (Invitrogen, Carlsbad, CA). Chicken HS4 (cHS4) insulator elements (1.2 kb) were cloned into the BamHI and SacI sites of pIRES2-EGFP from the plasmid DNA construct, pJC13-1 ([10]; kindly provided by Dr. Gary Felsenfeld, NIDDK, NIH). After successful transfer of two cHS4 insulator elements into pIRES2-EGFP, a DNA fragment encompassing the cHS4 insulator elements and partial MCS
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between the insulators was cloned into pKT2-meIF-SB100X or pKT2-RV SB vectors by appropriate combination of restriction enzyme digestions and modification of DNA ends with
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Klenow, T4 DNA polymerase and Antarctic Phosphatase (New England Biolabs, Tozier, MA), producing insulated SB vectors, pKT2/meIF-SB100X-Ins-MCS-Ins and pKT2/RV-Ins-MCSIns. Two insulators encompassing partial MCS were placed in the same, parallel direction to
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minimize loss of transgenes by recombination. As a final step to create SB constructs of IHK-
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β-globin transgene flanked by two cHS4 insulator elements, a 3.2-kb IHK-β-globin transgene fragment was derived from pT2/IHK-β-globin//eIF-SB10 [4] by digestion with PstI, and ligated into the partial MCS of pKT2/meIF-SB100X-Ins-MCS-Ins and pKT2/RV-Ins-MCSIns. The final constructs were termed pKT2/meIF-SB100X-IIβgI and pKT2/RV-IIβgI,
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respectively. Control SB-IHK-β-globin constructs with no insulator were made by cloning 3.2-kb IHK-β-globin transgene fragment into pKT2-meIF-SB100X and pKT2-RV vectors by
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EcoRV digestion and blunt-end ligations, resulting in pKT2/meIF-SB100X-Iβg and
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pKT2/RV-Iβg, respectively.
SB vectors of cHS4-insulated CAGGS-DsRed fluorescent transgene cassette were constructed with pKT2/meIF-SB100X-Ins-MCS-Ins and pKT2/RV-Ins-MCS-Ins in a similar manner. A CAGGS-DsRed transgene was derived from digestion of pCAGGS-DsRed with SpeI and HindIII, where DsRed transgene was obtained with PCR from pDsRed-Express (Clontech Laboratories, Inc., Mountain View, CA) and cloned into pCAGGS vector [16]. The
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CAGGS-DsRed transgene fragment was then blunted by Klenow (New England Biolabs) and cloned into SalI recognition site of pKT2/meIF-SB100X-Ins-MCS-Ins and pKT2/RV-Ins-
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MCS-Ins, making pKT2/meIF-SB100X-Ins-CAGGS-DsRed-Ins and pKT2/RV-Ins-CAGGSDsRed-Ins, respectively. Non-insulated control vectors, pKT2/meIF-SB100X-CAGGSDsRed and pKT2/RV-CAGGS-DsRed, were generated by directly cloning SpeI-HindIII
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CAGGS-DsRed transgene into original pKT2-meIF-SB100X and pKT2-RV, via blunt-end
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ligation.
Cell culture, transfection, and isolation of single-cell clones
K-562 cells (ATCC catalog #CCL-243) were cultured in RPMI 1640 (Invitrogen)
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supplemented with 10% FBS (Omega Scientific, Inc., Tarzana, CA) and 100 U/ml penicillin, 100 µg/ml streptomycin sulfate and 0.25 µg/ml amphotericin B. Lipofectamine™ 2000
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(Invitrogen) was used to transfect K-562 cells. 18 h prior to transfection, 1 × 106 cells were freshly plated on 100-mm Petri dishes in RPMI 1640 medium without antibiotics. 5 µg of
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plasmid DNA was used for each transfection according to the manufacturer’s instructions. Cells were once passaged in fresh medium after 18 h post-transfection, sorted using flow cytometry based on DsRed expression, and plated in 96-well plates to obtain single-cell clones. These clones were then transferred to 6-well plates and eventually maintained in 100mm plates for a period of 7-12 weeks. Isolation of single-cell clones for IHK-β-globin transgene [4] was carried out with
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pKT2/meIF-SB100X-IIβgI. Control SB-IHK-β-globin constructs with no insulator were also used to test whether cHS4 elements were effective in protecting the transgene from long-term
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silencing. Overall experimental procedures were similar to those for CAGGS-DsRed transgene transfer, except for flow cytometry. For IHK-β-globin clones, single-cell clones were isolated on a random basis, and later tested for the presence of SB-mediated insertion of
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IHK-β-globin transgene in the host genome by IHK-β-globin-specific PCR. At the indicated
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Isolation of genomic DNA
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time points, hemin (20 µM) was added 48 h prior to the cells being harvested.
Genomic DNA was isolated from ~ 5×106 K-562 cells using the DNeasy Blood & Tissue
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Kit according to manufacturer’s instructions (Qiagen).
Southern blot analysis
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Biotin-labeled probes were generated by PCR with biotin-16-dUTP (Roche Diagnostics
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Corp., Indianapolis, IN). A 100 µM aliquot of biotin-16-dUTP substituted dTTP (also 1:100 µM in the final PCR mixture) was added in a 1:1 ratio to make a final 200 µM of dTTP+dUTP in the PCR reaction. As templates for the PCR, SB vector constructs harboring necessary primer target area were used. Primers specific for DsRed transgene (DsR12) were: 5’-GAACGTCATCACCGAGTTCA-3’ and 5’-TAGTCCTCGTTGTGGGAGGT-3’; and primers for the β-globin transgene (BGprb) were: 5’-GAATGGGAAACAGACGAATGA-3’ and
5’-AATCCAGCCTTATCCCAACC-3’.
PCR
products
were
verified
by
gel-
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electrophoresis, and increase in the molecular weight of the probe products by the incorporation of biotin-dUTP was confirmed. Probe DNA molecules were then purified by
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gel-extraction. An amount of 15 to 20 µg of genomic DNA was digested by restriction enzyme (New England Biolabs) in a volume of 200 µl, and later concentrated by conventional ethanol precipitation to ~ 50 µl. When the second digestion with a different
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second digestion was carried out in a volume of 50 µl.
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restriction enzyme was required, DNA after the first digestion was purified to ~ 40 µl, and the
Digested genomic DNA was size-fractionated in an 8% agarose gel, and processed in denaturation buffers (1.5 M NaCl, 0.5 M NaOH) for 30 min. The gel was then incubated in the neutralization buffer (1.5 M NaCl, 0.5 M Tris·HCl, pH 7.4) for 30 min. DNA was
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subsequently transferred to Hybond-N+ nylon membrane by capillary blotting according to manufacturer’s instructions (GE Healthcare, Pittsburgh, PA). Immediately after the capillary
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transfer, DNA was fixed on the membrane by 70 mJ/cm2 of UV Stratalinker 2400 (Stratagene,
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Santa Clara, CA), and washed x 1 in 2×SSPE for 20 min. The membrane was then hybridized with biotin-labeled probes in PerfectHybTM Plus (Sigma-Aldrich, St. Louis, MO) hybridization solution as described in the manufacturer’s recommended protocol. Posthybridization washing was also performed according to the manufacturer’s protocol of PerfectHybTM Plus. After hybridization, the membrane was blocked in 1× TBS, 0.5% SDS, and 0.5% BSA for 30 min at room temperature. The membrane was then transferred to fresh
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blocking solution (50 ml) containing 1.25 µg of HRP-Conjugated Straptavidin (Thermo Fisher Scientific, Waltham, MA) for 1 h at room temperature. The membrane was washed 3
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times, each time for 5 min, with washing solution (1× TBS, 0.5% SDS), and the chemiluminescence reaction was carried out with Super Signal West Dura Chemiluminescent Substrate. The membrane was exposed to X-ray film for about 7 min.
Construction of SB-Tn vectors
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3. Results and discussion
To detect SB transposition in mammalian cells, we constructed vectors carrying units of DsRed cassettes with or without the cHS4 insulator element (Fig. 1A). When digested with
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KpnI restriction endonuclease, and detected by DsR12 probe indicated in the map, DsRed-SB integrants into the genome of K-526 cells were expected to produce DNA fragments larger
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than 2.8 kb with cHS4 element or larger than 1.6 kb without the cHS4 element.
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Similarly, vector constructs for the β-globin transgene with or without cHS4 were also created (Fig. 1B). When digested with BamHI, insulated (with cHS4) β-globin-SB produce DNA fragments larger than 3 kb, and non-insulated control β-globin-SB produced fragments larger than 1.8 kb, based on the nearest available restriction enzyme recognition site in the genomic area of SB insertion.
3.2
Detection of DsRed-containing SB transposons in the genome of K-562 cells by non-
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To detect the integration of DsRed-SB in K-562 cells, we generated single cell clones that
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were transfected with either insulated or non-insulated SB-DsRed constructs. These clones were subjected to non-radioactive Southern blot analyses. Clones 1, 3, 31, 32, 33 revealed more than a dozen insertions, some over 20 insertions in the genome of host cells (Fig. 2A).
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As predicted in the vector construct maps, minimum size of the DNA fragment signal was
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found to be > 3 kb. These results indicated that SB100X was indeed hyperactive, demonstrating a large number of SB integrations in the host genome of K-562 cells even when the cargo of SB was > 6 kb.
They also demonstrated that the SB insertions in host
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genomes could be detected by non-radioactive Southern blot analyses. Similar results were obtained with SB-Tn constructs without the cHS4 element (Fig. 2B).
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Most of the clones demonstrated a large number of insertions, emphasizing the hyperactivity of SB100X. When compared with Fig. 2A, the number of insertions seemed to be greater with
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non-insulated DsRed-SB, based on the size difference of SB constructs used to generate the clones (6 kb with two insulators and ~ 3.7 kb without the insulator). As predicted from the vector construct map (Fig. 1B), minimum size of the signal was over 1.6 kb. For these studies, genomic DNA isolated from K-562 cells was used as a negative control, whereas the positive control was genomic DNA from non-transfected K-562 cells that was mixed with DsRed-SB vector DNA in an amount equivalent to five copies in the genome. As expected, negative
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controls showed no specific signal because K-562 cells do not possess a DsRed transgene. However, when a 5-copy equivalent of DsRed-SB DNA was added to these cells (positive
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control), a strong signal of expected size was detected (Fig. 1B), excluding the possibility of cross-reaction of DsRed probes with host genomic DNA. These results further demonstrated that non-radioactive Southern blot analyses could be used as a tool to analyze and quantify
Detection of β-globin-containing SB transposons in the genome of K-562 cells by non-
radioactive Southern blot analyses
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3.3
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SB-insertion numbers by comparing the copy numbers in the host genome.
To further validate out non-radioactive Southern blot analysis, we carried out studies on SB
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integration by constructing SB-Tn vectors with IHK-β-globin transgene, with and without the cHS4 insulator element (Fig. 1B). The IHK-β-globin vector-specific signal was located
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between two smaller DNA fragments from endogenous β-globin locus. A series of clones were then generated by transfecting SB-Tn vectors into K-562 cells.
Southern blot analyses
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of these clones revealed the minimum size of inserted IHK-β-globin-SB with two insulator elements was ~ 3 kb depending on the insertion locus in the host chromosomes (Fig. 3A). Even though DNA fragments generated from endogenous β-globin gene were detected throughout different samples (in close comparison with SB negative control), most clones contained a very large number of SB insertions (in particular, clones 28, 30, 42, and 49), demonstrating the excessive activity of SB100X transposase. Furthermore, this result
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indicated that the previously reported significant limit on the SB cargo size was surpassed by the hyperactivity of SB100X because of the presence of a very large copy number of SB
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insertions even with 6-kb cargo. In these experiments, a negative control was genomic DNA isolated from K-562 cells, and the positive control was genomic DNA from non-transfected K-562 cells mixed with IHK-β-globin-SB vector DNA in an amount equivalent to 5 copies.
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Negative controls demonstrated a unique pattern of three DNA fragments most probably
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derived from the endogenous β-globin gene loci in the host K-562 cells. In contrast, the positive control revealed a distinctive signal due to the presence of vector DNA in addition to the endogenous β-globin gene (Fig. 3A).
When Southern blot analyses were carried out to
detect IHK-β-globin-SB insertions in K-562 clones created without the cHS4 element, we
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obtained similar results (Fig. 3B). The minimum size of DNA fragments was 1.8 kb, with many insertions of SB present. We further tested clones of 16 and 19 on two different types of
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membranes, and obtained an identical pattern of SB insertions (data not shown).
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Collectively, these results clearly showed that our non-radioactive Southern blot analyses were highly specific, and capable of detecting SB integrations in a reproducible manner.
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Methylation-sensitivity of K-562 clones containing SB-Tn insertions
Due to the potential for methylation within the CAGGS promoter of DsRed constructs, and IHK in β-globin constructs, we tested it using NarI restriction endonuclease, a commonly used enzyme for methylation-sensitive Southern blots [17]. For this purpose, one of the K-
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562 clones generated with insulated IHK-β-globin-SB was analyzed by sequential digestion first with SacI and then with NarI, whose recognition site is located at the IHK promoter (Fig.
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1B). If the NarI site was not methylated, it would be fully susceptible to NarI digestion causing the generation of 1.9-kb fragments. Indeed, this was the case with clone 33 (Fig. 4A). Numerous DNA fragments generated by SacI digestion (Fig. 4A, left lane) converged to a
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single band of 1.9 kb, clearly indicating that there were many SB insertions that were not
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methylated at the IHK promoter. However, almost half of the SacI-digested DNA fragments remained undigested by NarI (Fig. 4, SacI+NarI lane), implying that not all SB insertions in the clone 33 avoided CpG methylation. This result could be interpreted as partial protection of IHK promoter from negative epigenetic modification by cHS4 elements in the SB-Tn.
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To compare the DNA methylation patterns between insulated and non-insulated IHK-βglobin-SB constructs, a clone with non-insulated IHK-β-globin (#21) was also analyzed by
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non-radioactive Southern blot analysis using the combination of BglII and NarI enzymes (Fig.
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4A). As expected, none of the BglII-generated fragments (Fig. 4A, left lane) produced a 1.7kb signal, which represented the non-methylated SB insertions. These results indicated that non-radioactive methylation Southern blot could be useful for the investigation of SB methylation. A similar Southern blot approach was attempted for the investigation of DNA methylation at the CAGGS promoter of DsRed expression cassette of SB in the host genome. Combined
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digestion of clone #35 with SacI and NarI clearly showed that the SB insertions were almost completely protected from CpG methylation by cHS4 insulator elements, and most of the
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SacI-digested fragments were not visible (Fig. 4B, left lane) producing only 1.5-kb fragments when digested additionally with NarI (Fig. 4B, right lane, 1-35). On the other hand, a single clone of K-562 cell line harboring CAGGS-DsRed-SB without cHS4 insulator elements
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(clone #41) did not show any NarI-sensitivity (Fig. 4B). Even though this study confirmed
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the potential of methylation-sensitive non-radioactive Southern blot analysis, further studies
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are needed, where other enzymes can be tested for sensitivity to DNA methylation.
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Figure legends Fig. 1. Construction of SB-Tn vectors: (A) Schematic maps of SB transposon units containing DsRed transgene cassettes with (top) or without (bottom) the cHS4 insulator
without (bottom) cHS4 elements.
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element. (B) Vector constructs for the β-globin transgene were described with (top) or Recognition sites for NarI restriction endonuclease,
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which were used for the methylation-sensitive Southern blot, are denoted at the CAGGS
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promoter on the map.
Fig. 2. Non-radioactive Southern blot analyses of K-562 clones generated with DsRed containing SB-Tn constructs (shown in Figure 1A).
(A) Southern blotting was performed
DsRed.
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to detect SB-Tn containing DsRed in K-562 clones that were generated with insulated SBGenomic DNA from K-562 clones 1, 3, 5, 27-29, and 31-33 clones were digested After
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with KpnI, and the digested fragments were separated by gel electrophoresis.
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transferring the fragments onto Hybond-N+ nylon membranes, they were hybridized with biotin-labeled probes against DsRed (see Materials and Methods). (B) Southern blotting was performed to detect the SB-Tn integrated into the genome of K-562 clones 3-5, 8-12, 29,30, 32, 33, 35, 36, 38 and 40. These clones were generated with the SB-Tn containing DsRed, but not the cHS4 insulator element. Genomic DNA from each clone was digested with KpnI, and Southern blot assay was performed as in (A). SB (–) is the genomic DNA isolated from K-
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562 cells without any modification (negative control). SB (+) is genomic DNA from nontransfected K-562 cells, mixed with DsRed-SB vector construct DNA in an amount
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equivalent to 5 copies in the genome (positive control).
Fig. 3. Non-radioactive Southern blot analyses of K-562 clones generated with IHK-β(A) Southern blotting was
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globin containing SB-Tn constructs (shown in Fig. 1B).
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performed to detect SB-Tn containing DsRed in K-562 clones that were generated with insulated SB-IHK-β-globin. Genomic DNA from K-562 clones 28, 30 and 49 was digested with BamHI, and that of 30-35, 40, 42, 47, and 48 was digested with SacI. SB (-) is the genomic DNA isolated from K-562 cells without any modification. SB (+) control is the
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genomic DNA from non-transfected K-562 cells, mixed with IHK-β-globin-SB vector construct DNA in an amount equivalent to 5 copies in the genome. (B) Southern blotting was
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performed to detect SB-Tn containing DsRed in K-562 clones that were generated with non-
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insulated SB-IHK-β-globin. Experimental procedures were as described in Materials and Methods, and under Fig. 2.
Fig. 4. Methylation-sensitive non-radioactive Southern blot analysis.
(A) To explore
the possibility of methylation-sensitivity of K-562 clones, non-radioactive Southern blot analysis was performed with one clone each of insulated (cHS4) (#33), and non-insulated
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IHK-β-globin-SB was analyzed by sequential digestion first with SacI and
secondly with methylation-sensitive restriction enzyme NarI, of which recognition site is
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located at the IHK promoter (Fig. 1B) for clone #33; BglII and BglII+NarI digestion was performed with clone 21. (B) A similar Southern blot approach was attempted for the investigation of DNA methylation at the CAGGS promoter of DsRed expression cassette of
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SB in the host genome, using insulated (#35) and uninsulated (#41) clones, respectively.
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[11] C.L. Li, D. W. Emery, The cHS4 chromatin insulator reduces gammaretroviral vector
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silencing by epigenetic modifications of integrated provirus, Gene Ther. 15 (2008) 49– 53.
[12] M.J. Pikaart, F. Recillas-Targa, G. Felsenfeld, Loss of transcriptional activity of a transgene is accompanied by DNA methylation and histone deacetylation and is prevented by insulators, Genes Dev. 12 (1998) 2852–2862. [13] T. Dalsgaard, B. Moldt, N. Sharma, G. Wolf, A. Schmitz, F.S. Pedersen, J.G. Mikkelsen,
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Shielding of Sleeping Beauty DNA transposon-delivered transgene cassettes by heterologous insulators in early embryonal cells., Mol. Ther. 17 (2009) 121–130.
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[14] O. Walisko, A. Schorn, F. Rolfs, A. Devaraj, C. Miskey, Z. Izsvák, Z. Ivics, Transcriptional activities of the Sleeping Beauty transposon and shielding its genetic cargo with insulators, Mol. Ther. 16 (2008) 359–369.
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[15] L.M. Sjeklocha, C.W. Park, P.Y. Wong, M.J. Roney, J.D. Belcher, D.S. Kaufman, G.M.
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Vercellotti, R.P. Hebbel, C.J. Steer, Erythroid-specific expression of β-globin from Sleeping Beauty-transduced human hematopoietic progenitor cells, PLoS ONE 6 (2011) e29110.
[16] H. Niwa, K. Yamamura, J. Miyazaki, Efficient selection for high-expression
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transfectants with a novel eukaryotic vector, Gene 108 (1991) 193–199. [17] T. Moore, Southern analysis using methyl-sensitive restriction enzymes, Methods Mol.
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Biol. 181 (2001) 193–203.
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S0006-291X(16)31012-9
DOI:
10.1016/j.bbrc.2016.06.094
Reference:
YBBRC 36010
To appear in:
Biochemical and Biophysical Research Communications
Received Date: 3 June 2016 Accepted Date: 18 June 2016
Please cite this article as: R.N. Aravalli, C.W. Park, C.J. Steer, Detection of Sleeping Beauty transposition in the genome of host cells by non-radioactive Southern blot analysis, Biochemical and Biophysical Research Communications (2016), doi: 10.1016/j.bbrc.2016.06.094. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Detection of Sleeping Beauty transposition in the genome of host cells by non-radioactive Southern blot analysis
a
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Rajagopal N. Aravalli a,b,1, *,¶, Chang W. Park b,1, and Clifford J. Steer b,c,*
Department of Radiology, University of Minnesota Medical School, MMC 292, 420
b
Department of Medicine, University of Minnesota Medical School, MMC 36, 420 Delaware
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Street SE, Minneapolis, MN 55455, USA c
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Delaware Street SE, Minneapolis, MN 55455, USA
Department of Genetics, Cell Biology and Development, University of Minnesota,
Minneapolis, MN 55455, USA
These authors contributed equally to the work.
*
Corresponding authors
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¶
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E-mail: [email protected]; [email protected]
Present address: Department of Electrical and Computer Engineering, University of
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Minnesota, 200 Union Street SE, Minneapolis, MN 55455, USA
Number of pages: 28 (including the figures) Number of figures: 4
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Abstract The Sleeping Beauty transposon (SB-Tn) system is being used widely as a DNA vector for the delivery of therapeutic transgenes, as well as a tool for the insertional mutagenesis in animal
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models. In order to accurately assess the insertional potential and properties related to the integration of SB it is essential to determine the copy number of SB-Tn in the host genome. Recently developed SB100X transposase has demonstrated an integration rate that was much
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higher than the original SB10 and that of other versions of hyperactive SB transposases, such as HSB3 or HSB17. In this study, we have constructed a series of SB vectors carrying either a
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DsRed or a human β-globin transgene that was encompassed by cHS4 insulator elements, and containing the SB100X transposase gene outside the SB-Tn unit within the same vector in cis configuration. These SB-Tn constructs were introduced into the K-562 erythroid cell line, and their presence in the genomes of host cells was analyzed by Southern blot analysis using nonradioactive probes. Many copies of SB-Tn insertions were detected in host cells regardless of
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transgene sequences or the presence of cHS4 insulator elements. Interestingly, the size difference of 2.4 kb between insulated SB and non-insulated controls did not reflect the proportional difference in copy numbers of inserted SB-Tns. We then attempted methylation-
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sensitive Southern blots to assess the potential influence of cHS4 insulator elements on the
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epigenetic modification of SB-Tn. Our results indicated that SB100X was able to integrate at multiple sites with the number of SB-Tn copies larger than 6 kb in size. In addition, the nonradioactive Southern blot protocols developed here will be useful to detect integrated SB-Tn copies in any mammalian cell type.
Keywords: Epigenetic; Methylation; Sleeping Beauty; SB100X; Southern blot; Transposon
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Introduction Sleeping Beauty (SB) is a member of the Tc1/Mariner superfamily of DNA transposons
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that are widespread in nature, but are transpositionally inactive in vertebrates due primarily to the accumulation of mutations [1]. SB is a synthetic transposon, which was originally constructed based on sequences of transpositionally inactive elements found in fish genomes
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[2]. The resurrected SB was engineered by removing the inactivating mutations from the
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transposase open reading frame. By using high throughput genetic screening of SB variants, a hyperactive version SB100X, that displayed ~ 100-fold higher activity than the originally resurrected transposase, was identified [3]. This SB transposon (SB-Tn) system yielded robust gene transfer efficiencies in a variety of mammalian cells, and enabled highly efficient
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germline transgenesis in relevant animal models of human diseases (reviewed in [1]). We previously reported DNA methylation in both IHK promoter and DsRed transgene
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sequences, which was correlated with the level of IHK-DsRed transgene expression in the
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host genome of K-562 cells after integration via SB-Tn [4] . DNA methylation of transgenes inserted into the host genome has been reported in other studies using retroviral vectors [5,6], which led to progressive DNA methylation and transgene silencing. CpG methylation was also reported in transgenes delivered and integrated into animal cells by SB-Tn systems [4,7,8]. Studies that were able to correlate the expression of transgenes delivered via the SBTn system with the level of DNA methylation demonstrated a predicted inverse relationship
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with expression [4,7]. In an attempt to prevent this epigenetic silencing of the transgenes by DNA methylation, we introduced heterologous chicken HS4 β-globin insulator elements
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(cHS4) into our SB transgene cassettes. This HS4 insulator element was able to block enhancer activity [9,10], thereby either eliminating or diminishing the influence of enhancer elements on genes in close proximity, whether the enhancer was included in the exogenous
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transgene constructs or placed close to the inserted exogenous transgene in the host genome.
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In addition, HS4 insulator is known to protect transgenes from silencing by epigenetic modification, such as CpG methylation when the transgene is shielded by cHS4 insulator elements [11,12]. In a recent report, it was shown that successful expression of the fluorescent transgene can be maintained for long term when the transgene was flanked by the
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cHS4 insulator element in a SB-Tn vector [13]. Furthermore, we could expect the enhancer blocking activity of the insulator to prohibit the potential transactivation of neighboring host
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genes by SB-mediated transgene insertion, which we observed in a few insertion loci [4,14].
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Therefore, we cloned cHS4 insulator elements into our SB vector constructs so that they flank both sides of the transgene cassette within SB transposons defined by IR/DR sequences. Control vectors, which lack just the insulator element, were also generated and tested in comparison with insulated SB constructs. To achieve maximum efficiency of transposon insertion into the host genome, we used SB100X [3]. For all our SB-Tn vectors, whether in cis- or trans-configuration, SB100X was
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the source of transposase, and the expression of the transgenes was initially tested in an erythroid cell line, K-562. Using non-radioactive probes in Southern blotting, we successfully
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detected SB transposons in host cells. As expected from the hyperactive SB transposase SB100X, a large number of insertions of SB transgene cassettes were detected, of which some were greater than 6 kb in size. Additionally, we also confirmed a possibility of assessing
2. Materials and methods
Sleeping Beauty transposon constructs
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2.1
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sensitive non-radioactive Southern blot analyses.
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DNA methylation status of SB constructs containing cHS4 elements by using methylation
The cis pKT2-meIF-SB100X and trans pKT2-RV SB-Tn vectors [15] contained the
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hyperactive SB100X transposase gene driven by the mouse eukaryotic initiation factor 4A1 promoter (InVivogen, San Diego, CA). We first made SB constructs in which SB transposon
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units had two insulator elements (cHS4; [10]) flanking multiple restriction endonuclease recognition sites (MCS) derived from pIRES2-EGFP (Invitrogen, Carlsbad, CA). Chicken HS4 (cHS4) insulator elements (1.2 kb) were cloned into the BamHI and SacI sites of pIRES2-EGFP from the plasmid DNA construct, pJC13-1 ([10]; kindly provided by Dr. Gary Felsenfeld, NIDDK, NIH). After successful transfer of two cHS4 insulator elements into pIRES2-EGFP, a DNA fragment encompassing the cHS4 insulator elements and partial MCS
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between the insulators was cloned into pKT2-meIF-SB100X or pKT2-RV SB vectors by appropriate combination of restriction enzyme digestions and modification of DNA ends with
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Klenow, T4 DNA polymerase and Antarctic Phosphatase (New England Biolabs, Tozier, MA), producing insulated SB vectors, pKT2/meIF-SB100X-Ins-MCS-Ins and pKT2/RV-Ins-MCSIns. Two insulators encompassing partial MCS were placed in the same, parallel direction to
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minimize loss of transgenes by recombination. As a final step to create SB constructs of IHK-
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β-globin transgene flanked by two cHS4 insulator elements, a 3.2-kb IHK-β-globin transgene fragment was derived from pT2/IHK-β-globin//eIF-SB10 [4] by digestion with PstI, and ligated into the partial MCS of pKT2/meIF-SB100X-Ins-MCS-Ins and pKT2/RV-Ins-MCSIns. The final constructs were termed pKT2/meIF-SB100X-IIβgI and pKT2/RV-IIβgI,
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respectively. Control SB-IHK-β-globin constructs with no insulator were made by cloning 3.2-kb IHK-β-globin transgene fragment into pKT2-meIF-SB100X and pKT2-RV vectors by
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EcoRV digestion and blunt-end ligations, resulting in pKT2/meIF-SB100X-Iβg and
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pKT2/RV-Iβg, respectively.
SB vectors of cHS4-insulated CAGGS-DsRed fluorescent transgene cassette were constructed with pKT2/meIF-SB100X-Ins-MCS-Ins and pKT2/RV-Ins-MCS-Ins in a similar manner. A CAGGS-DsRed transgene was derived from digestion of pCAGGS-DsRed with SpeI and HindIII, where DsRed transgene was obtained with PCR from pDsRed-Express (Clontech Laboratories, Inc., Mountain View, CA) and cloned into pCAGGS vector [16]. The
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CAGGS-DsRed transgene fragment was then blunted by Klenow (New England Biolabs) and cloned into SalI recognition site of pKT2/meIF-SB100X-Ins-MCS-Ins and pKT2/RV-Ins-
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MCS-Ins, making pKT2/meIF-SB100X-Ins-CAGGS-DsRed-Ins and pKT2/RV-Ins-CAGGSDsRed-Ins, respectively. Non-insulated control vectors, pKT2/meIF-SB100X-CAGGSDsRed and pKT2/RV-CAGGS-DsRed, were generated by directly cloning SpeI-HindIII
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CAGGS-DsRed transgene into original pKT2-meIF-SB100X and pKT2-RV, via blunt-end
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ligation.
Cell culture, transfection, and isolation of single-cell clones
K-562 cells (ATCC catalog #CCL-243) were cultured in RPMI 1640 (Invitrogen)
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supplemented with 10% FBS (Omega Scientific, Inc., Tarzana, CA) and 100 U/ml penicillin, 100 µg/ml streptomycin sulfate and 0.25 µg/ml amphotericin B. Lipofectamine™ 2000
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(Invitrogen) was used to transfect K-562 cells. 18 h prior to transfection, 1 × 106 cells were freshly plated on 100-mm Petri dishes in RPMI 1640 medium without antibiotics. 5 µg of
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plasmid DNA was used for each transfection according to the manufacturer’s instructions. Cells were once passaged in fresh medium after 18 h post-transfection, sorted using flow cytometry based on DsRed expression, and plated in 96-well plates to obtain single-cell clones. These clones were then transferred to 6-well plates and eventually maintained in 100mm plates for a period of 7-12 weeks. Isolation of single-cell clones for IHK-β-globin transgene [4] was carried out with
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pKT2/meIF-SB100X-IIβgI. Control SB-IHK-β-globin constructs with no insulator were also used to test whether cHS4 elements were effective in protecting the transgene from long-term
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silencing. Overall experimental procedures were similar to those for CAGGS-DsRed transgene transfer, except for flow cytometry. For IHK-β-globin clones, single-cell clones were isolated on a random basis, and later tested for the presence of SB-mediated insertion of
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IHK-β-globin transgene in the host genome by IHK-β-globin-specific PCR. At the indicated
2.3
Isolation of genomic DNA
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time points, hemin (20 µM) was added 48 h prior to the cells being harvested.
Genomic DNA was isolated from ~ 5×106 K-562 cells using the DNeasy Blood & Tissue
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Kit according to manufacturer’s instructions (Qiagen).
Southern blot analysis
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Biotin-labeled probes were generated by PCR with biotin-16-dUTP (Roche Diagnostics
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Corp., Indianapolis, IN). A 100 µM aliquot of biotin-16-dUTP substituted dTTP (also 1:100 µM in the final PCR mixture) was added in a 1:1 ratio to make a final 200 µM of dTTP+dUTP in the PCR reaction. As templates for the PCR, SB vector constructs harboring necessary primer target area were used. Primers specific for DsRed transgene (DsR12) were: 5’-GAACGTCATCACCGAGTTCA-3’ and 5’-TAGTCCTCGTTGTGGGAGGT-3’; and primers for the β-globin transgene (BGprb) were: 5’-GAATGGGAAACAGACGAATGA-3’ and
5’-AATCCAGCCTTATCCCAACC-3’.
PCR
products
were
verified
by
gel-
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electrophoresis, and increase in the molecular weight of the probe products by the incorporation of biotin-dUTP was confirmed. Probe DNA molecules were then purified by
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gel-extraction. An amount of 15 to 20 µg of genomic DNA was digested by restriction enzyme (New England Biolabs) in a volume of 200 µl, and later concentrated by conventional ethanol precipitation to ~ 50 µl. When the second digestion with a different
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second digestion was carried out in a volume of 50 µl.
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restriction enzyme was required, DNA after the first digestion was purified to ~ 40 µl, and the
Digested genomic DNA was size-fractionated in an 8% agarose gel, and processed in denaturation buffers (1.5 M NaCl, 0.5 M NaOH) for 30 min. The gel was then incubated in the neutralization buffer (1.5 M NaCl, 0.5 M Tris·HCl, pH 7.4) for 30 min. DNA was
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subsequently transferred to Hybond-N+ nylon membrane by capillary blotting according to manufacturer’s instructions (GE Healthcare, Pittsburgh, PA). Immediately after the capillary
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transfer, DNA was fixed on the membrane by 70 mJ/cm2 of UV Stratalinker 2400 (Stratagene,
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Santa Clara, CA), and washed x 1 in 2×SSPE for 20 min. The membrane was then hybridized with biotin-labeled probes in PerfectHybTM Plus (Sigma-Aldrich, St. Louis, MO) hybridization solution as described in the manufacturer’s recommended protocol. Posthybridization washing was also performed according to the manufacturer’s protocol of PerfectHybTM Plus. After hybridization, the membrane was blocked in 1× TBS, 0.5% SDS, and 0.5% BSA for 30 min at room temperature. The membrane was then transferred to fresh
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blocking solution (50 ml) containing 1.25 µg of HRP-Conjugated Straptavidin (Thermo Fisher Scientific, Waltham, MA) for 1 h at room temperature. The membrane was washed 3
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times, each time for 5 min, with washing solution (1× TBS, 0.5% SDS), and the chemiluminescence reaction was carried out with Super Signal West Dura Chemiluminescent Substrate. The membrane was exposed to X-ray film for about 7 min.
Construction of SB-Tn vectors
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3. Results and discussion
To detect SB transposition in mammalian cells, we constructed vectors carrying units of DsRed cassettes with or without the cHS4 insulator element (Fig. 1A). When digested with
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KpnI restriction endonuclease, and detected by DsR12 probe indicated in the map, DsRed-SB integrants into the genome of K-526 cells were expected to produce DNA fragments larger
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than 2.8 kb with cHS4 element or larger than 1.6 kb without the cHS4 element.
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Similarly, vector constructs for the β-globin transgene with or without cHS4 were also created (Fig. 1B). When digested with BamHI, insulated (with cHS4) β-globin-SB produce DNA fragments larger than 3 kb, and non-insulated control β-globin-SB produced fragments larger than 1.8 kb, based on the nearest available restriction enzyme recognition site in the genomic area of SB insertion.
3.2
Detection of DsRed-containing SB transposons in the genome of K-562 cells by non-
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To detect the integration of DsRed-SB in K-562 cells, we generated single cell clones that
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were transfected with either insulated or non-insulated SB-DsRed constructs. These clones were subjected to non-radioactive Southern blot analyses. Clones 1, 3, 31, 32, 33 revealed more than a dozen insertions, some over 20 insertions in the genome of host cells (Fig. 2A).
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As predicted in the vector construct maps, minimum size of the DNA fragment signal was
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found to be > 3 kb. These results indicated that SB100X was indeed hyperactive, demonstrating a large number of SB integrations in the host genome of K-562 cells even when the cargo of SB was > 6 kb.
They also demonstrated that the SB insertions in host
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genomes could be detected by non-radioactive Southern blot analyses. Similar results were obtained with SB-Tn constructs without the cHS4 element (Fig. 2B).
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Most of the clones demonstrated a large number of insertions, emphasizing the hyperactivity of SB100X. When compared with Fig. 2A, the number of insertions seemed to be greater with
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non-insulated DsRed-SB, based on the size difference of SB constructs used to generate the clones (6 kb with two insulators and ~ 3.7 kb without the insulator). As predicted from the vector construct map (Fig. 1B), minimum size of the signal was over 1.6 kb. For these studies, genomic DNA isolated from K-562 cells was used as a negative control, whereas the positive control was genomic DNA from non-transfected K-562 cells that was mixed with DsRed-SB vector DNA in an amount equivalent to five copies in the genome. As expected, negative
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controls showed no specific signal because K-562 cells do not possess a DsRed transgene. However, when a 5-copy equivalent of DsRed-SB DNA was added to these cells (positive
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control), a strong signal of expected size was detected (Fig. 1B), excluding the possibility of cross-reaction of DsRed probes with host genomic DNA. These results further demonstrated that non-radioactive Southern blot analyses could be used as a tool to analyze and quantify
Detection of β-globin-containing SB transposons in the genome of K-562 cells by non-
radioactive Southern blot analyses
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SB-insertion numbers by comparing the copy numbers in the host genome.
To further validate out non-radioactive Southern blot analysis, we carried out studies on SB
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integration by constructing SB-Tn vectors with IHK-β-globin transgene, with and without the cHS4 insulator element (Fig. 1B). The IHK-β-globin vector-specific signal was located
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between two smaller DNA fragments from endogenous β-globin locus. A series of clones were then generated by transfecting SB-Tn vectors into K-562 cells.
Southern blot analyses
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of these clones revealed the minimum size of inserted IHK-β-globin-SB with two insulator elements was ~ 3 kb depending on the insertion locus in the host chromosomes (Fig. 3A). Even though DNA fragments generated from endogenous β-globin gene were detected throughout different samples (in close comparison with SB negative control), most clones contained a very large number of SB insertions (in particular, clones 28, 30, 42, and 49), demonstrating the excessive activity of SB100X transposase. Furthermore, this result
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indicated that the previously reported significant limit on the SB cargo size was surpassed by the hyperactivity of SB100X because of the presence of a very large copy number of SB
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insertions even with 6-kb cargo. In these experiments, a negative control was genomic DNA isolated from K-562 cells, and the positive control was genomic DNA from non-transfected K-562 cells mixed with IHK-β-globin-SB vector DNA in an amount equivalent to 5 copies.
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Negative controls demonstrated a unique pattern of three DNA fragments most probably
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derived from the endogenous β-globin gene loci in the host K-562 cells. In contrast, the positive control revealed a distinctive signal due to the presence of vector DNA in addition to the endogenous β-globin gene (Fig. 3A).
When Southern blot analyses were carried out to
detect IHK-β-globin-SB insertions in K-562 clones created without the cHS4 element, we
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obtained similar results (Fig. 3B). The minimum size of DNA fragments was 1.8 kb, with many insertions of SB present. We further tested clones of 16 and 19 on two different types of
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membranes, and obtained an identical pattern of SB insertions (data not shown).
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Collectively, these results clearly showed that our non-radioactive Southern blot analyses were highly specific, and capable of detecting SB integrations in a reproducible manner.
3.4
Methylation-sensitivity of K-562 clones containing SB-Tn insertions
Due to the potential for methylation within the CAGGS promoter of DsRed constructs, and IHK in β-globin constructs, we tested it using NarI restriction endonuclease, a commonly used enzyme for methylation-sensitive Southern blots [17]. For this purpose, one of the K-
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562 clones generated with insulated IHK-β-globin-SB was analyzed by sequential digestion first with SacI and then with NarI, whose recognition site is located at the IHK promoter (Fig.
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1B). If the NarI site was not methylated, it would be fully susceptible to NarI digestion causing the generation of 1.9-kb fragments. Indeed, this was the case with clone 33 (Fig. 4A). Numerous DNA fragments generated by SacI digestion (Fig. 4A, left lane) converged to a
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single band of 1.9 kb, clearly indicating that there were many SB insertions that were not
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methylated at the IHK promoter. However, almost half of the SacI-digested DNA fragments remained undigested by NarI (Fig. 4, SacI+NarI lane), implying that not all SB insertions in the clone 33 avoided CpG methylation. This result could be interpreted as partial protection of IHK promoter from negative epigenetic modification by cHS4 elements in the SB-Tn.
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To compare the DNA methylation patterns between insulated and non-insulated IHK-βglobin-SB constructs, a clone with non-insulated IHK-β-globin (#21) was also analyzed by
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non-radioactive Southern blot analysis using the combination of BglII and NarI enzymes (Fig.
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4A). As expected, none of the BglII-generated fragments (Fig. 4A, left lane) produced a 1.7kb signal, which represented the non-methylated SB insertions. These results indicated that non-radioactive methylation Southern blot could be useful for the investigation of SB methylation. A similar Southern blot approach was attempted for the investigation of DNA methylation at the CAGGS promoter of DsRed expression cassette of SB in the host genome. Combined
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digestion of clone #35 with SacI and NarI clearly showed that the SB insertions were almost completely protected from CpG methylation by cHS4 insulator elements, and most of the
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SacI-digested fragments were not visible (Fig. 4B, left lane) producing only 1.5-kb fragments when digested additionally with NarI (Fig. 4B, right lane, 1-35). On the other hand, a single clone of K-562 cell line harboring CAGGS-DsRed-SB without cHS4 insulator elements
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(clone #41) did not show any NarI-sensitivity (Fig. 4B). Even though this study confirmed
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the potential of methylation-sensitive non-radioactive Southern blot analysis, further studies
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are needed, where other enzymes can be tested for sensitivity to DNA methylation.
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Figure legends Fig. 1. Construction of SB-Tn vectors: (A) Schematic maps of SB transposon units containing DsRed transgene cassettes with (top) or without (bottom) the cHS4 insulator
without (bottom) cHS4 elements.
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element. (B) Vector constructs for the β-globin transgene were described with (top) or Recognition sites for NarI restriction endonuclease,
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which were used for the methylation-sensitive Southern blot, are denoted at the CAGGS
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promoter on the map.
Fig. 2. Non-radioactive Southern blot analyses of K-562 clones generated with DsRed containing SB-Tn constructs (shown in Figure 1A).
(A) Southern blotting was performed
DsRed.
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to detect SB-Tn containing DsRed in K-562 clones that were generated with insulated SBGenomic DNA from K-562 clones 1, 3, 5, 27-29, and 31-33 clones were digested After
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with KpnI, and the digested fragments were separated by gel electrophoresis.
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transferring the fragments onto Hybond-N+ nylon membranes, they were hybridized with biotin-labeled probes against DsRed (see Materials and Methods). (B) Southern blotting was performed to detect the SB-Tn integrated into the genome of K-562 clones 3-5, 8-12, 29,30, 32, 33, 35, 36, 38 and 40. These clones were generated with the SB-Tn containing DsRed, but not the cHS4 insulator element. Genomic DNA from each clone was digested with KpnI, and Southern blot assay was performed as in (A). SB (–) is the genomic DNA isolated from K-
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562 cells without any modification (negative control). SB (+) is genomic DNA from nontransfected K-562 cells, mixed with DsRed-SB vector construct DNA in an amount
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equivalent to 5 copies in the genome (positive control).
Fig. 3. Non-radioactive Southern blot analyses of K-562 clones generated with IHK-β(A) Southern blotting was
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globin containing SB-Tn constructs (shown in Fig. 1B).
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performed to detect SB-Tn containing DsRed in K-562 clones that were generated with insulated SB-IHK-β-globin. Genomic DNA from K-562 clones 28, 30 and 49 was digested with BamHI, and that of 30-35, 40, 42, 47, and 48 was digested with SacI. SB (-) is the genomic DNA isolated from K-562 cells without any modification. SB (+) control is the
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genomic DNA from non-transfected K-562 cells, mixed with IHK-β-globin-SB vector construct DNA in an amount equivalent to 5 copies in the genome. (B) Southern blotting was
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performed to detect SB-Tn containing DsRed in K-562 clones that were generated with non-
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insulated SB-IHK-β-globin. Experimental procedures were as described in Materials and Methods, and under Fig. 2.
Fig. 4. Methylation-sensitive non-radioactive Southern blot analysis.
(A) To explore
the possibility of methylation-sensitivity of K-562 clones, non-radioactive Southern blot analysis was performed with one clone each of insulated (cHS4) (#33), and non-insulated
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IHK-β-globin-SB was analyzed by sequential digestion first with SacI and
secondly with methylation-sensitive restriction enzyme NarI, of which recognition site is
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located at the IHK promoter (Fig. 1B) for clone #33; BglII and BglII+NarI digestion was performed with clone 21. (B) A similar Southern blot approach was attempted for the investigation of DNA methylation at the CAGGS promoter of DsRed expression cassette of
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SB in the host genome, using insulated (#35) and uninsulated (#41) clones, respectively.
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References [1]
Z. Izsvák, Z. Ivics, Sleeping Beauty transposition., Microbiol. Spectr. 3 (2015)
[2]
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MDNA3-0042–2014. Z. Ivics, P.B. Hackett, R.H. Plasterk, Z. Izsvák, Molecular reconstruction of Sleeping Beauty, a Tc1-like transposon from fish, and its transposition in human cells, Cell 91
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robust stable gene transfer in vertebrates, Nat. Genet. 41 (2009) 753–761. J. Zhu, C.W. Park, L. Sjeklocha, B.T. Kren, C.J. Steer, High-level genomic integration,
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B.S. Garrison, S. R. Yant, J. G. Mikkelsen, M.A. Kay, Postintegrative gene silencing
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[10] J.H. Chung, M. Whiteley, G. Felsenfeld, A 5' element of the chicken β-globin domain
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serves as an insulator in human erythroid cells and protects against position effect in Drosophila, Cell 74 (1993) 505–514.
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[11] C.L. Li, D. W. Emery, The cHS4 chromatin insulator reduces gammaretroviral vector
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Biol. 181 (2001) 193–203.
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1.5 kb DsRed
cHS4
SacI
CAGGS
probe DsR12 KpnI
cHS4
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Figure 1A
NarI, Methylation-sensitive
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1 kb
DsRed
CAGGS
IR/DR
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NarI, Methylation-sensitive
probe KpnI DsR12 BglII
IR/DR
TIR(R)
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IR/DR (L)
SacI
IHK
cHS4
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Human β-globin
cHS4
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1.9 kb
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Figure 1B
probe βGprb22 BamHI
IR/DR (R)
NarI, Methylation-sensitive
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1.7 kb
Human β-globin
IR/DR
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probe BglII βGprb22 BamHI
IHK
IR/DR NarI, Methylation-sensitive
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Figure 2A
KpnI 5
27 28 29 31 32 33
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3
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1
KpnI
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KpnI digestion
KpnI digestion
40
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32 33 35 36 38
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29 30
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Figure 2B
3 4 5 8
9 10 11 12
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Figure 3A
BamHI
SacI
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49 28 30
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30 31 32 34 35 42 47 48 49
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Figure3B
BamHI
BamHI
19
4 11 14 15 16 19 20 71
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B
A #21
#35
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#33
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Figure 4
#41