Ribozyme and siRNA oligonucleotides
Six genes of single ribozymes zb1, zb2, zb3, zb4, zb5, and zb6 against human βS-globin mRNA were synthesized with a 22-bp core sequence, as zb1, TGTTTGAGGTTGCTAGTGctgatgagtccgtgaggacgaaACACAGTTGTGTCAGAAG; zb2, ACAGGGCAGTAACGGCActgatgagtccgtgaggacgaaACTTCTCCTCAGGAGTCA; zb3, GCCCAGGGCCGCCACCACCctgatgagtccgtgaggacgaaACTTCATCCACTTCACC; zb4, AGATCCCCAAAGctgatgagtccgtgaggacgaaACTCAAAGAACCTC; zb5, TTAGGGTTGCCCATctgatgagtccgtgaggacgaaACAGCATCAGGA; and zb6, TGGGCCAGCACACActgatgagtccgtgaggacgaaACCAGCCGTTGCC; and their presumed cleavage sites on human βS-globin mRNA are shown in . In order to facilitate ligation in later steps, restriction site pairs Kpn I/Xho I, Xho I/Cla I, Cla I/EcoR I, EcoR I/BamH I, BamH I/ Eag I, and Eag I/ Sac I were added to both ends of the synthetic oligonucleotides zb1-zb6, respectively.
Expected ribozyme cleavage sites.
Three siRNA genes were synthesized as hairpin structures with a 9-bp loop, as ib2, GGTGAACGTGGATGAAGTTGGTttcgaaagaACCAACTTCATCCACGTTCACC; ib4, GGTGAAGGCTCATGGCAAGAAAttcgaaagaTTTCTTGCCATGAGCCTTCACC; and ib5, GAAAGTGCTCGGTGCCTTTAGTGATttcgaaagaATCACTAAAGGCACCGAGCACTTTC corresponding to β-globin mRNA 104-125, 230-251, and 248-272, respectively.
The oligonucleotides were synthesized as sense and antisense chains using Applied Biosystems 308 DNA Synthesizer (Applied Biosystems, Foster City, CA) and annealed for ligation.
Plasmids for producing ribozymes and mRNA substrates in vitro
Six annealed ribozyme oligonucleotides were inserted into pBluescript II (Stratagene, La Jolla, CA) to form plasmids pKSzb1, pKSzb2, pKSzb3, pKSzb4, pKSzb5, and pKSzb6. The 0.47-kb NgoM IV/Xho I fragment containing the ribozyme zb2 from pKSzb2 was inserted into the 2.59-kb NgoM IV/Xho I vector immediately ahead of the ribozyme zb1 gene to form plasmid pKSzb21, which contained the multi-ribozyme gene zb21 which consisted of tandemly arranged ribozyme genes zb2 and zb1, linked by a Xho I site. In a similar manner, the other four ribozyme genes, zb6, zb5, zb4, and zb3, were stepwise added on pKSzb21, and finally plasmid pKSzb61 which contained multi-ribozyme zb61, composed of tandemly arranged ribozyme genes, zb6, zb5, zb4, zb3, zb2, and zb1, was constructed. In addition, plasmid pKSzb21A containing multi-ribozyme zb21A was constructed from pKSzb21 by replacing the Xho I site between the zb2 and zb1 sequences with an antisense βS-globin mRNA fragment (GGTGCACCATGCATGGTGTC). All these single and multi-ribozyme genes in plasmids described above were located between the T7 and T3 promoter and derived from plasmid pBluescript II.
Plasmid pKShac’ containing the T7 promoter directed human α-globin cDNA was described previously [32
]. Plasmids pKShbSc’ and pKShgc’ containing the T7 promoter directed human βS
-globin cDNA and γ-globin cDNA were constructed by respectively placing human β-globin cDNA fragment from pHE7 and Aγ-globin cDNA fragment from pHE9 [37
] into pBluescript II after the T7 promoter. A β6 Glu→Val mutation [19
] was introduced into β-globin cDNA to produce human βS
-globin cDNA in pKShbSc’.
Plasmids for ribozyme expression in K562 cells
The human βS-globin gene fragment (exon 1-IVS1-exon 2-IVS2-exon 3) excluding the promoter was engineered into an NcoI/Pst1 fragment (1268 bp) and inserted downstream of the human cytomegalovirus (CMV) immediate early promoter in the pSP64 vector (Promega, Madison, IL) to make a pCMV-βS construct. The neomycin resistant gene in plasmid pCMV-βS was replaced by a hygromycin resistant gene, and the 0.49-kb Hind III/BamH I fragment of its βS-globin gene was replaced by single and multi-ribozyme genes zb1-zb6, zb21A, and zb61 to form plasmids pHC-zb1-pHC-zb6, pHC-zb21A, and pHC-zb6, in which the corresponding ribozyme and multi-ribozyme genes were directed by the CMV promoter. A similar plasmid with no ribozyme gene insertion was constructed as pHC-0. In addition, a 1.68-kb luciferase gene fragment from pGL3-control (Promega) was inserted into plasmid pCMV-βS after codons 18 and 56 of the βS-globin gene. The resultant plasmids pNC-βS18:luc and pNC-βS56:luc, respectively, contained a βS-globin N-terminal 1-18 and 1-56 amino acid coding sequence fused with a complete luciferase gene directed by the CMV promoter.
Plasmids for ribozyme, long hairpin β-globin mRNA and siRNA expression in progenitor cells
Plasmid pHLG-zb21A containing the multi-ribozyme zb21A gene directed by a shortened human locus control region (LCR) of the β-globin gene cluster and γ-globin promoter was constructed by modifying plasmid pKLCRa2-Zc [32
] in following manner: (i) HS1 fraction of LCR was deleted; (ii) α2-globin promoter was replaced by Gγ-globin promoter (-728 - -1); (iii) Zc gene was replaced by multi-ribozyme gene zb21A; and (iv) neomycin resistant gene was replaced by hygromycin resistant gene from pTKhyg (BD Biosciences, Palo Alto, CA). The control plasmid pHLG-0 was constructed by deleting the zb21A gene of pHLG-zb21A. Plasmids pCA-0 and pCA-zb21A are similar to pHC-0 and pHC-zb21A, except the hygromycin resistant gene was deleted, and the LCR-Gγ globin promoter was replaced by CMV promoter.
The long hairpin β-globin mRNA expression plasmid pHC-Fbi1 was constructed from pHC-zb21A, in which the zb21A portion was replaced by a hairpin sequence corresponding to β-globin cDNA 1-133, with a 26-bp loop.
siRNA expression plasmids, pEU-ib2, pEU-ib4, and pEU-ib5, were constructed by inserting corresponding siRNA gene ib2, ib4, and ib5 (see Ribozyme and siRNA Oligonucleotides
) into plasmid pSilencer2.0-U6 (Ambion, Austin, TX) between the U6 promoter and TTTTTGGAA sequence [38
] and a 0.40-kb enhancer sequence of CMV immediate early promoter (27-431 in pCMVβ, BD Biosciences, Palo Alto, CA) was placed before the U6 promoter [39
In-vitro transcription and ribozyme cleavage assay
32P-labeled human α-, βS-, and γ-globin mRNAs were respectively prepared by in-vitro transcription using MAXIscript T7 kit (Ambion) with [α-32P]-UTP as the labeled substrate (800 Ci/mmole, Amersham Pharmacia Biotech, Piscataway, NJ) with Spe I linearized pKShac’ and Xho I linearized pKShbSc’ and pKShgc’ as templates. Unlabeled single and multi-ribozyme RNA zb1-zb6, zb21A, and zb61 were prepared using the same kit with Acc65 I linearized pKSzb1-pKSzb6, pKSzb21A and pKSzb61 as templates. Antisense zb21A RNA was synthesized using a MAXIscript T3 kit (Ambion) with Hind III linearized pKSzb21A as template. All in-vitro transcription products were purified with a RNeasy mini kit (Qiagen, Valencia, CA) and their concentrations were estimated by the total isotope incorporation or by UV absorption.
ribozyme cleavage reaction has been described previously [32
], except that we used incubation times of 20 and 60 min.
Cell culture and transfection
K562βS cells are derived from human erythroleukemia cells K562 by transfection of the human βS-globin expression plasmid pCMV-βS. For stable integration into K562 cells, the vector contained a neomycin resistant gene cassette and was transfected into K562 cells, and stable clones were selected using G418 (Invitrogen Corp., Carlsbad, CA). Stable expression of βS-globin was confirmed by RT-PCR and sequencing of the reaction product to confirm the presence of the βS-globin mutation. The expression level of βS-globin mRNA in K562βS cells is 391±0.9 attomole/μg total RNA.
The conditions of K562βS
cell culture, stable transfection, and separation of single cell colonies have been described previously [32
]. Plasmid DNAs used in transfection were prepared using QIAwell 8 plasmid kits (Qiagen). The cells were cultured in RPMI media with no added antibiotics right before transfection.
K562 cells were used in the transient transfection experiment, and LipofectAMINE 2000 (Invitrogen) was used as the transfection reagent. The transient transfection procedure followed the manufacturer’s protocol. Stable transfection and single-cell colony culture have been described previously [32
], except we used hygromycin (200 μg /ml) as the selection reagent.
Primer extension assay
Total RNA was prepared from transfected cells or single-cell colonies using RNeasy mini kits (Qiagen). Total mRNA was prepared from total RNA using GenElute mRNA Miniprep Kit (Sigma, St Louis, MO).
Relative amounts of cellular βS
-globin mRNA, ribozyme RNA, and other mRNAs in the K562βS
cells were determined by primer extension assay. 5′-32
P-labeled oligonucleotide primers complementary to βS
-globin mRNA, α-globin mRNA, γ-globin mRNA, β-actin mRNA, and ribozyme RNAs, respectively (see Supplemental Data, Table 1S
) were prepared with [γ-32
P]-ATP (>5000Ci/mmole, Amersham Pharmacia Biotech) and T4 polynucleotide kinase (New England Biolabs, Beverly MA) reaction, and were purified with a QIAquick nucleotide removal kit (Qiagen). We added 0.1 - 0.5 pmole of 32
P-primers to reaction mixtures containing total RNA sample, 1 M NaCl, 0.2 M tris-HCl (pH 7.0), and 0.1 M EDTA in a total volume of 100 μl. The mixtures were incubated at 70°C for 3 min and then at 55°C for 1 hr. Then, 0.1 volume of 3 M NaOAc and 2 volumes of ethanol were added, and the mixtures were placed at -20°C for at least 1 hr. The annealed 32
P-primer-RNA products were precipitated, washed and then dissolved in 11 μl H2
O. We then added 4 μl 5× first stranded buffer containing 15 mM MgCl2
(Invitrogen), 1 μl dNTP (10 mM), 1 μl DTT (0.1 M), 1 μl RNsin (39 units, Promega), and 2 μl MMLV reverse transcriptase (400 units, Invitrogen). The extension reaction proceeded at 42°C for 1 hr. To stop the reaction, we added 20 μl loading buffer II (Ambion), the mixtures were heated at 95°C for 3 min., placed in 0°C bath for 3 min, and then loaded on a 5% polyacrylamide gel with 8 M urea. After electrophoresis, the radioactive bands corresponding to different mRNAs or ribozyme RNAs were located by autoradiography, and the radioactivity of each band was determined by direct counting of the bands that had been cut from the gel using a Beckman LS 7000 scintillation counter. The relative amount of βS
-globin mRNA, ribozyme RNA, and other mRNAs was calculated from the radioactivity data using β-actin mRNA as an internal standard. In order to simplify the calculation, 32
P-primers for the different RNA tests were labeled with the same specific activity, except for βS
-globin mRNA. The γ/βS
ratio was directly calculated by the ratio of relative amount of γ-globin mRNA and βS
βS-globin mRNA and other RNAs were also determined by primer extension using total mRNA samples. In such tests, because both βS-globin mRNA and the expressed ribozyme existed in the samples, excess degradation of βS-globin mRNA by the co-existed ribozyme may happen. We have found that such degradation can be completely inhibited by adding 0.1 μM antisense-ribozyme RNA into the annealing mixture before primer extension (results not shown). The antisense zb21A RNA can be easily prepared by in-vitro transcription with T3 promoter using pKSzb21A as template.
βS-Globin:luciferase fusion protein assay
βS-globin18:luciferase and βS-globin56:luciferase fusion proteins produced in K562 cells transfected with pNC-βS18:luc and pNC-βS56:luc were directly detected in transient transfection cell lysates using luminometer (Perkin-Elmer Victor2 1420 luminometer, Wellesley, MA) with Bright-Glo reagent (Promega) following the manufacturer’s protocol. The cell lysate transfected by luciferase expression plasmid pGL3-control was used as a transfection efficiency control.
Hematopoietic progenitor cell cultures
Blood was obtained from consenting normal volunteers from the NIH Department of Transfusion Medicine and hematopoietic progenitor cells were harvested and grown in liquid culture [40
]. Mononuclear cells were isolated by centrifugation on Ficoll-Hypaque (BioWhittaker, Walkersville, MD). Cells were cultured in α-minimal essential medium supplemented with 10% fetal bovine serum (FBS) (both from Gibco, Grand Island, NY), 10% conditioned medium from bladder carcinoma 5637 cultures, 1.5 mM glutamine (Biofluids, Rockville, MD), 1 μg/ml cyclosporin A (Sigma Chemical Co., St. Louis, MO), and antibiotics. After 5-7 days, non-adherent cells were washed twice with Dulbecco’s phosphate buffered saline without Ca+2
, and transferred to erythropoietin containing medium which consisted of α-minimal essential medium supplemented with 30% FBS (both from Gibco), 1% deionized bovine serum albumin, 10-6
M dexamethasone, 10-5
M β-mercapthoethanol, 0.3 mg/ml human hollo-transferrin (all from Sigma Chemical Co.), 10 ng/ml human recombinant stem cell factor (PeproTech, Rocky Hill, NJ), 1 U/ml human recombinant erythropoietin (EPO) (Amgen Inc., Thousand Oaks, CA), and antibiotics. Cultures were incubated at 37°C in an atmosphere of 5% CO2
and 100% humidity in standard incubators. On day 1 of culture with EPO stimulation, cells were transfection by electroporation with plasmids containing either a multi-ribozyme gene, a long hairpin β-globin cDNA gene, or a siRNA gene. Electroporation was performed with the Amaxa electroporation system (Amaxa, Gaithersburg, MD) using their human CD34 cell nucleofector kit, following the included protocol.
Cells were harvested and mRNA analyzed on day 12 of culture following EPO stimulation. First-strand cDNA was synthesized from 1 mg of total RNA using MuLV reverse transcriptase (RT) and oligo-d(T)16
(Applied Biosystems, Foster City, CA). Quantitative real-time RT-PCR was used to determine the level of mRNA expression with gene-specific primers and fluorescent labeled Taqman probes on a 7700 Sequence Detector (Applied Biosystems) [41
]. Probes were designed to span exon junctions in order to prevent the amplification of any contaminating genomic DNA and were fluorescently labeled with FAM (6-carboxy-fluorescein) as the 5′-fluorescent reporter and TAMERA (6-carboxy-tetramethyl-rhodamine) as the 3′ end quencher. Probes and primers were generated using Primer Express (Applied Biosystems). PCR reaction conditions were 50°C for 2 min, 95°C for 4 min and 40 cycles of 95°C (melting temperature) for 15 sec and 60°C (annealing-extension temperature) for 1 min. At low amplification, threshold cycle number (Ct) is directly proportional to the amount of corresponding specific mRNA. Standard curves were created using serial dilutions of plasmids containing the cDNA of interest. Human β-actin was used to normalize all results.