We present the results of a thorough study of wet chemical methods for transferring chemical vapor deposition grown graphene from the metal growth substrate to a device-compatible substrate. On the basis of these results, we have developed a "modified RCA clean" transfer method that has much better control of both contamination and crack formation and does not degrade the quality of the transferred graphene. Using this transfer method, high device yields, up to 97%, with a narrow device performance metrics distribution were achieved. This demonstration addresses an important step toward large-scale graphene-based electronic device applications.

We have mapped the distribution of the major and minor DNase I-hypersensitive sites in the human "fi-like-globin" gene domain. The minor DNase I-hypersensitive sites map close to the 5' end of each of the 13-like-globin genes. Their presence is specifically associated with the transcription of the immediate downstream fi-like-globin genes.The major DNase I-hypersensitive sites map in what appear to be the 5' and 3' boundary areas of the human fi-like-globin gene domain, a region estimated to span at least 90 kilobases of DNA. These major sites are present in various erythroid cells, which express predominantly either the embryonic, the fetal, or the adult (3-like-globin genes, and seem to be involved in derming the active j8-like-globin gene domain in cells of erythroid lineage. The four major DNase I-hypersensitive sites in the 5' boundary area, when correlated with sequencing data, are shown to be located in DNA regions containing enhancer core-like sequences and alternating purine and pyrimidine bases.The human "/3-like-globin" genes (hemoglobin P-chain gene cluster) encode, respectively, one embryonic (e), two fetal (G y and Ay), and two adult (8 and /3) globin chains. These genes have been shown to reside within "50 kilobases (kb) of chromosomal DNA in the transcriptional order 5' e-y.GAy. EXPERIMENTAL PROCEDURES Cells were grown as described (7). Human bone marrow cells were collected from cancer patients with normal marrow who were to undergo chemotherapy and bone marrow reinfusion. Isolated by dextran column chromatography, -25% of the nucleated cells were erythroid.DNase I-digestion, gel electrophoresis, RNA isolation, blotting, and hybridization were carried out as described (7). RESULTS Globin Gene Transcription in K562,HEL, Adult Human Marrow, and HL60 Cells. Nuclear and cytoplasmic RNAs were isolated from cells, and individual globin gene transcription was detected by "dot-blot" hybridization with e-,

No abstract

No abstract

We have previously described the development of oncoretrovirus vectors for human ␥-globin using a truncated ␤-globin promoter, modified ␥-globin cassette, and ␣-globin enhancer. However, one of these vectors is genetically unstable, and both vectors exhibit variable expression patterns in cultured cells, common characteristics of oncoretrovirus vectors for globin genes. To address these problems, we identified and removed the vector sequences responsible for genetic instability and flanked the resultant vector with the chicken ␤-globin HS4 chromatin insulator to protect expression from chromosomal position effects. After determining that flanking with the cHS4 element allowed higher, more uniform levels of ␥-globin expression in MEL cell lines, we tested these vectors using a mouse bone marrow transduction and transplantation model. When present, the ␥-globin cassettes from the uninsulated vectors were expressed in only 2% to 5% of red blood cells (RBCs) long term, indicating they are highly sensitive to epigenetic silencing. In contrast, when present the ␥-globin cassette from the insulated vector was expressed in 49% ؎ 20% of RBCs long term. RNase protection analysis indicated that the insulated ␥-globin cassette was expressed at 23% ؎ 16% per copy of mouse ␣-globin in transduced RBCs. These results demonstrate that flanking a globin vector with the cHS4 insulator increases the likelihood of expression nearly 10-fold, which in turn allows for ␥-globin expression approaching the therapeutic range for sickle cell anemia and ␤ thalassemia. IntroductionThe ␤ chain hemoglobinopathies ␤ thalassemia and sickle cell anemia constitute the most common class of hereditary, monogenic disorders in the human population, affecting hundreds of thousands of persons worldwide. 1 In ␤ thalassemia, a lack of ␤-globin synthesis results in the precipitation of free ␣-globin chains and the subsequent destruction of erythroid precursors in the marrow. 1 In sickle cell anemia, a single amino acid substitution in the ␤-globin chain leads to globin chain polymerization, red cell sickling, and subsequent vascular occlusions and red cell destruction. 2 Recent therapeutic interventions include the use of cytotoxic drugs to induce the synthesis of fetal ␥-globin, which can bind up free ␣-globin chains in ␤-thalassemia 3,4 and can interfere with globin chain polymerization in sickle cell anemia. [5][6][7] However, these agents have proven ineffective for the treatment of severe transfusion-dependent ␤ thalassemia, and safety concerns remain about the lifelong administration of cytotoxic drugs in patients with sickle cell disease. Allogeneic bone marrow transplantation can cure patients with ␤ chain hemoglobinopathies. 1,8,9 However, this procedure is limited by the availability of HLA-identical donors and morbidity and mortality risks that increase as the clinical phenotype of these diseases worsens with age. For these reasons, we and others have pursued the development of gene therapy for the treatment of the ␤ chain hemoglobinopathies.The...