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6 cells per milliliter in BIT 9500 serum-free medium (Stem Cell Technology) supplemented with Flt-3 ligand (25 ng/ml) (Immunex), IL-3 (2.5 ng/ml) (R&D), and erythropoietin (1 unit/ml) (R&D). These cells were then cultured at 37°C overnight and transferred to fresh plates to eliminate adherent cells.
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2642684167
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note
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The mapping library was generated by PCR amplification of plasmid pT7L-21 (7) with a 5′ primer containing a randomized sequence at positions corresponding to the ribozyme's IGS (5′-GGGGG-GATCCTAATACGACTCACTATAGNNNNNAAAAG TTATCAGGCATGCACC) and a 3′ primer specific for 3′ exon tag sequences present in the pT7L-21 plasmid (5′-AGTAGTCTTACTGCAGGGGCCTCTTCGCTATTACG). The resulting cDNA library was in vitro transcribed using T7 RNA polymerase to generate the RNA mapping library.
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21
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2642599649
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note
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6 cells) were resuspended in 200 μl of Opti-MEM (Gibco-BRL), and ribozymes (2.5 to 5 μg generated by in vitro transcription) were transfected into these cells using 20 μl of DMRIE-C (Gibco-BRL) in 1 ml of Opti-MEM for 4 hours. Then, DMEM (Gibco-BRL) with 10% fetal calf serum (1 ml) and erythropoietin (2 units/ml) were added to the cells. Total RNA was isolated by using TRI Reagent (Molecular Research Center) containing EDTA (10 mM) 16 to 24 hours after transfection. Transfection of these cells with a reporter RNA demonstrated that 1 to 2% of the erythrocyte precursors take up RNA (17).
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22
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2642627276
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note
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2] and then equilibrated at 37°C for 3 min. The substrates were then added to the ribozymes along with guanosine (100 μM) to start the reactions, which proceeded at 37°C for 3 hours. For reaction mixtures that contained radiolabeled ribozyme, we removed samples at the times indicated and added them to an equal volume of EDTA (10 mM) to stop the reaction. Reaction products were analyzed on a 4% polyacrylamide gel containing 8 M urea. For RT-PCR analysis, trans-splicing products were reverse transcribed at 37°C for 20 min in the presence of L-argininamide (10 mM) from a primer specific for the 3′ exon sequence as described (8, 9). The resulting cDNAs were amplified for 30 cycles (in vitro ribozyme reactions) or 30 to 90 cycles (in vivo ribozyme reactions) using a 3′ exon primer (3′tag primer, 5′-ATGCCTGCAGGTCGACTC; 3′γ-globin primer, 5′-CCGGAATTCCCTTGTCCTCCTCTGTGA) (9, 20) and a 5′ primer specific for the β-globin mRNA (5′-GGG-GATCCCTGTGTTCACTAGCAACC). The amplified products were separated on a 10% acrylamide gel and visualized by ethidium bromide staining.
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23
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2642695322
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unpublished results
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N. Lan, unpublished results.
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Lan, N.1
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25
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2642589624
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note
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S-globin is spliced, RNA is reduced from 161 to 111 bp. Unreacted globin substrate RNAs as well as revised β-globin products were coamplified by using a single set of PCR primers to yield different-sized products that were separated on an acrylamide gel, and Phosphorlmager analysis was used to quantify the efficiency with which the ribozyme had converted the globin RNA to product in the reaction (18).
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26
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2642657954
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note
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5, however, because the activity of the free ribozymes appears to be suppressed by the excess of unreacted substrate present in the sample (17). The observation that the longer substrates do not appear to suppress this miscleavage as well as their shorter counterpart suggests that not all the longer RNAs are folded into a conformation that allows for ribozyme binding. In the long run, ribozymes with increased substrate specificity will be developed to solve this miscleavage problem.
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28
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0017182027
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2642603830
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note
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We thank C. Rusconi, P. Zarrinkar, L. Milich, J. Jones, K. Phillips, M. Barman, R. Kaufman, M. Telen, E. Gilboa, H. Lyerly, and R. Rempel for useful discussions, M. Telen for her generous gift of the E6 antibody, and the nurses in labor and delivery and the medical oncology treatment center for their assistance in collecting blood samples. Supported in part by the Korean Academic Research Fund of the Ministry of Education (S.-W.L.) and by a grant (HL57606) from the National Heart Lung and Blood Institute (B.A.S.).
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