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-,

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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...

The human ␤-globin locus contains five actively transcribed genes that are arranged in their developmental order of expression. High-level expression of the ␤-globin gene cluster is dependent on the presence of the locus control region (LCR) (18), an element characterized by a series of five DNase I-hypersensitive sites (HSs) located 6 to 22 kb upstream of the ε-globin gene (9,10,18,44). Naturally occurring deletions of this element result in changes in chromatin structure that extend at least 200 kb 3Ј of the deletion, transcriptional silencing of the ␤-globin locus, and a phenotype of ␤ thalassemia (4,5,12,22). Functional properties of the LCR include activation of the ␤-globin locus (10, 18), restriction of globin gene expression to cells of the erythroid cell lineage (18, 45), enhancement of globin gene expression (11,18,39), and protection from position effects of globin genes transferred in transgenic mice (13,18,25,41).Transgenic mice have been extensively used to study the developmental control of the ␤-globin genes, the function of the LCR, and the role of individual HSs in ␤-globin gene regulation. Linkage of individual HSs to individual globin genes have shown that HS2, HS3, and HS4 are capable of conferring position-independent expression of globin genes, with stronger activation of expression at a specific stage of development (14, 25). Several observations have led to a model suggesting that the HSs form a complex that directly interacts with globin gene promoters by looping of the intervening DNA (7, 28, 46). HS2, HS3, and HS4 have 200-to 400-bp core regions that are able to provide position-independent expression in transgenic mice (27,34,35,37,42). These HS core regions may be indispensable components of the LCR complex; deletions of the HS3 or HS4 core elements result in disruption of HS function and reduction of globin gene expression (3).Discernment of the function of individual HSs and analysis of how the LCR interacts with individual genes during development require studies in the context of intact, native ␤-globin loci. Entire ␤-globin loci have been used to generate transgenic mice, by ligating two cosmids to produce a 70-kb fragment (40) or by using 248-kb (30) or 150-kb (15, 36) yeast artificial chromosomes harboring the ␤-globin locus (␤-YACs). Mice carrying ␤-YACs show correct regulation of the human globin genes, presumably because all the human cis-regulatory elements are present in the transferred sequences of the ␤-globin locus and are properly recognized by the murine transacting environment. In ␤-YAC transgenic mice, the ε-globin gene is expressed during the embryonic stage of development and is confined to primitive erythropoiesis in the yolk sac. The ␥-globin genes are also expressed in the embryonic yolk sac, but unlike their murine homologous gene, ␤h1, ␥-globin gene expression continues in the fetal liver stage of erythropoiesis. Human ␤-globin gene expression occurs only in the cells of definitive erythropoiesis.To delineate the role of HS3 in LCR function and globin gene ...

The use of redox-active molecules as the active storage elements in memory chips requires the ability to attach the molecules to an electroactive surface in a reliable and robust manner. To explore the use of porphyrins tethered to silicon via carbosilane linkages, 17 porphyrins have been synthesized. Fourteen porphyrins bear a tether at a single meso site, and three porphyrins bear functional groups at two beta sites for possible two-point attachment. Two high-temperature processing methods (400 degrees C under inert atmosphere) have been developed for rapid (minutes), facile covalent attachment to Si platforms. The high-temperature processing conditions afford attachment either by direct deposition of a dilute solution (1 microM-1 mM) of the porphyrin sample onto the Si substrate or sublimation of a neat sample onto the Si substrate. The availability of this diverse collection of porphyrins enables an in-depth examination of the effects of the tether (length, composition, terminal functional group, number of tethers) and steric bulk of nonlinking substituents on the information-storage properties of the porphyrin monolayers obtained upon attachment to silicon. Attachment proceeds readily with a wide variety of hydrocarbon tethers, including 2-(trimethylsilyl)ethynyl, vinyl, allyl, or 3-butenyl directly appended to the porphyrin and iodo, bromomethyl, 2-(trimethylsilyl)ethynyl, ethynyl, vinyl, or allyl appended to the 4-position of a meso-phenyl ring. No attachment occurs with substituents such as phenyl, p-tolyl, mesityl, or ethyl. Collectively, the studies show that the high-temperature attachment procedure (1) has broad scope encompassing diverse functional groups, (2) tolerates a variety of arene substituents, and (3) does not afford indiscriminate attachment. The high-temperature processing conditions are ideally suited for use in fabrication of hybrid molecular/semiconductor circuitry.

Identification of functional, noncoding elements that regulate transcription in the context of complex genomes is a major goal of modern biology. Localization of functionality to specific sequences is a requirement for genetic and computational studies. Here, we describe a generic approach, quantitative chromatin profiling, that uses quantitative analysis of in vivo chromatin structure over entire gene loci to rapidly and precisely localize cis-regulatory sequences and other functional modalities encoded by DNase I hypersensitive sites. To demonstrate the accuracy of this approach, we analyzed B300 kilobases of human genome sequence from diverse gene loci and cleanly delineated functional elements corresponding to a spectrum of classical cis-regulatory activities including enhancers, promoters, locus control regions and insulators as well as novel elements. Systematic, highthroughput identification of functional elements coinciding with DNase I hypersensitive sites will substantially expand our knowledge of transcriptional regulation and should simplify the search for noncoding genetic variation with phenotypic consequences.Understanding the human genome will require comprehensive delineation of functional elements within the 98% of genomic terrain that does not encode protein. In vivo, cis-regulatory modalities colocalize with focal alterations in chromatin structure [1][2][3][4] , and this governs the accessibility of genomic sequences to critical regulatory factors. Exploitation of the close connection between functional elements and chromatin structure should offer a powerful and generic approach for de novo identification of cis-regulatory sequences in the context of complex gene domains.Active regulatory elements within complex genomes are distinguished by pronounced sensitivity to the nonspecific endonuclease DNase I 3-5 when exposed in the context of intact nuclei. DNase I hypersensitive sites are the sine qua non of a diverse spectrum of classical transcriptional and chromosomal regulatory activities including enhancers, promoters, silencers, insulators, boundary elements and locus control regions 1,3,6 . Indeed, in the human genome, many functional elements were first identified as major DNase I hypersensitive sites and only later were found to have specific regulatory roles. Analysis of chromatin structure may enable generic delineation of functional elements across the genome, provided it exhibits direct sequence specificity, quantitative data output that permits automated analysis, and adaptability to a high-throughput format.DNase I hypersensitive sites in native genomic domains have traditionally been localized by an approach relying on Southern transfer followed by indirect end-labeling 5 . Although widely applied, this technique is not quantitative and has numerous technical and resource-related limitations that prevent its application on a genome-wide scale. The major limitations of conventional hypersensitivity assays are the low throughput and the lack of sequence specificity. Conventional So...