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De-Pei Liu

Publications and source records attributed to De-Pei Liu.

17 recordsLinked to original sources
PubMed2003

A hypothesis for chromatin domain opening.

The eukaryotic genome is organized into different domains by cis-acting elements, such as boundaries/insulators and matrix attachment regions, and is packaged with different degrees of condensation. In the M phase, the chromatin becomes further highly condensed into chromosomes. The first step for transcriptional activation of a given gene, at a particular time during development, in any locus, is the opening of its chromatin domain. This locus needs to be kept in this state in each early G(1) phase during every cell cycle. Certain distal enhance elements, including locus control regions (LCRs) and enhancers, are believed to perform this target chromatin domain opening process and several models have been proposed to explain distal enhance action. But they did not explain precisely how a given chromatin domain is opened. Based on various studies, we propose a hypothesis for the mechanism of opening chromatin on a large scale. One important mechanism may involved breaking one or two DNA strands and reducing the linking numbers within chromatin domain. The topological changes can overpass some complexes formed on DNA strands and can be transmitted from specific localized points over a broad region, until boundary elements or insulators are reached. These may initiate downstream events such as propagation of histone acetylation and the binding of transcription factors to proximal promoters and may further augment the action mediated by distal enhancer elements.

Chromatin↗
PubMed2003

Finding regulatory sequences.

DNA regulatory sequences control gene expression by forming DNA-protein complex with specific DNA binding protein. A major task of studies of gene regulation is to identify DNA regulatory sequences in genome-wide. Especially with the rapid pace of genome project, the function of DNA regulatory sequences becomes one of the focuses in functional genome era. Several approaches for screening and characterizing DNA regulatory sequences emerged one by one, from initial low-throughput methods to high-throughput strategies. Even though at present bioinformatics tools facilitate the process of screening regulatory fragments, the most reliable results will come from experimental test. This article highlights some experimental methods for the identification of regulatory sequences. A brief review of the history and procedures for selection methods are provided. Tendency as well as limitation and extension of these methods are also presented.

DNA↗
PubMed2003

MicroRNAs: key participants in gene regulatory networks.

microRNAs (miRNAs) are a newly identified and surprisingly large class of endogenous tiny regulatory RNAs. They exhibit various expressional patterns and are highly conserved across species. Recently, several regulatory targets of miRNAs have been predicted. Functional analysis of the potential targets indicated that miRNAs may be involved in a wide range of pivotally biological events. The nature of miRNAs and their intersection with small interfering RNAs endow them with many regulatory advantages over proteins and make them a potent and novel means to regulate gene expression at almost all levels. Here we argue that miRNAs are key participants in gene regulatory network.

Animals↗
PubMed2003

Evaluation of optimal expression cassette in retrovirus vector for beta-thalassemia gene therapy.

Trials of retroviral vector-mediated human beta-globin gene transfer were hampered by low titers, unstable vector transmission, and low-level expression of transferred gene. With the goal of optimizing the retrovirally encoded human beta-globin gene expression cassette for gene therapy of beta-thalassemia, we generated 3 series of vector constructs (a total of 12 constructs) and investigated the effects of the proximal promoter, 3' - enhancer, and derivatives from the beta-locus control region or alpha-major regulatory element on virus titer, vector transmission stability, and gene expression. The virus titers for 9 of the 12 vector constructs ranged between 2.8 x 10(4) cfu/mL and 1.0 x 10(6) cfu/mL. We found that proviral DNA was intact in most G418- resistant murine erythroleukemia (MEL) cell clones for 5 vector constructs, while obvious genetic instability was observed for 4 other vector constructs. MEL cells harboring the intact provirus were induced to differentiate, and human beta-globin gene expression was analyzed with RNase protection assay. The percentage of human beta-globin transcript relative to endogenous murine alpha-globin transcript were 101.8 +/- 64.3% (n = 10), 40.1 +/- 28.7% (n = 4), 31.1 +/- 31.9% (n = 12), 52.4 +/- 11.2% (n = 12), and 53.6 +/- 8.6% (n = 12) for the 5 constructs, respectively, demonstrating the development of optimized retroviral vectors for beta-globin gene therapy with murine erythroid cell lines as a model. Unexpectedly, we also documented that the point mutation 8700(C-->T) in DNase I hypersensitive site 2 (HS2) core fragment might contribute to low-level expression of the human beta-globin gene, based on a comparison of results from transfected and transduced MEL cells and sequence analysis of proviral DNA.

Animals↗
PubMed2003

Excessive expression of the scavenger receptor class A type I can significantly affect the serum lipids.

Scavenger receptor (SR) is characterized by its ability to bind negatively charged macromolecules, particularly the modified lipoproteins that are pertinent to the development of vascular disease. To determine the role of excessive scavenger receptor A in the serum lipoprotein metabolism, transgenic mice lines with mouse scavenger receptor A gene type I (SR-AI) under the control of human SR-AI enhancer and metallothionein gene promotor were established. After zinc induction, the expression of SR-AI in transgenic mice was a little higher than the controls, but the serum lipids levels were significantly different from the controls, especially the cholesterol. These results demonstrated that overexpression of SR-AI significantly affected the serum lipids levels.

Animals↗
PubMed2003

[Development of gene therapy for hematopoietic stem cell using viral vectors].

Hematopoietic stem cells (HSC) are attractive targets for gene therapy of inherited and acquired disorders in hematopoietic system in that they possess the properties of self-renewal, proliferation, and multi-lineage differentiation. For successful gene therapy, the viral vector-mediated gene addition strategy has two essential prerequisites: 1) the efficient transfer of therapeutic gene into HSC; 2) the long-term and stable expression of the transgene at therapeutic levels. The oncoretrovirus-derived vectors are best understood and most widely investigated. Recent successful cases of gene therapy for severe combined immunodeficiency due to adenosine deaminase or gamma c chain deficiencies have provided strong evidences that retrovirus-mediated gene transfer into HSC will work in clinical treatment. While these results are encouraging, some obstacles remain to be circumvented including low efficiency of gene transfer and gene silencing in retroviral vector system. The therapeutic gene can be efficiently introduced into HSC by HIV-1-based lentiviral vector due to its capability to infect the quiescent cells. A variety of preclinical studies are now conducted and a number of valuable results highlight the efficacy of lentiviral-mediated gene transfer into HSC. However, the potential value of lentiviral vectors in human gene therapy remains to be demonstrated. Adeno-associated virus vector is an alternative to retroviral and lentiviral vectors. This review summarizes the characteristics of integrating vectors, the improved HSC transduction protocols, and the optimized gene expression strategies and outlines the important advances of preclinical and clinical trials in hematopoietic stem cell gene therapy.

Adenoviridae↗
PubMed2002

Oligonucleotide-mediated gene repair at DNA level: the potential applications for gene therapy.

Mutations in gene sequence can cause many genetic disorders, and researchers have attempted to develop treatments or cures at the DNA level for these diseases. Several strategies including triple-helix-forming oligonucleotides (TFOs), chimeric RNA/DNA oligonucleotide (RDO), and short single-stranded oligodeoxynucleotide (ODN) have been used to correct the dysfunctional genes in situ in the chromosome. Experimental data from cells and animal models suggest that all these strategies can repair the mutations in situ at DNA level. More effective structures of oligonucleotide, efficient delivery systems, and gene correction efficiency should be improved. Development of these strategies holds great potentials for treatments of genetic defects and other disorders.

Animals↗
PubMed2002

Chromatin structure and transcriptional regulation of the beta-globin locus.

Chromatin structure plays a critical role in eukaryotic gene transcriptional regulation. The beta-globin locus provides an ideal system within which to study the interplay between chromatin structure and transcriptional regulation. The process of beta-globin locus activation is remarkably intricate and involves at least two distinct events: chromatin opening and gene activation. Great progress has been made in recent years in understanding how locus control regions confer high-level expression to linked genes. Current interest focuses on some special events, including formation of locus control region hypersensitivity sites, ATP-dependent chromatin remodeling, localized H3 hyperacetylation, and intergenic transcription, which link chromatin and beta-globin locus regulation. These events, and their possible molecular bases, are summarized together with speculations concerning their connections.

Acetylation↗
PubMed2002

Efficient isolation of regulatory sequences from human genome and BAC DNA.

Isolation of regulatory DNA fragments is the basis of the identification of DNA binding proteins and the study of the regulation of gene expression. Presently there is a lack of efficient methods to broadly isolate and identify DNA regulatory fragments. We developed an efficient method to isolate regulatory DNA sequences from both genome and bacterial artificial chromosome (BAC) based on electrophoretic mobility shift assay and PCR techniques without purified transcription factors. Twenty-nine DNA fragments were isolated from human genome and 24 from BAC DNA containing human apolipoprotein AI gene cluster. Transient transfection assay showed that some fragments could enhance the transcription of reporter gene.

Apolipoprotein A-I↗
PubMed2002

The control of expression of the alpha-globin gene cluster.

The alpha-globin gene cluster is located at the very tip of the short arm of chromosome 16. It produces the alpha-like globins, which is combined with the beta-like globins to form hemoglobin, and its mutants cause alpha-thalassemia, which is one of the most common genetic diseases. Its expression shows a tissue and developmental stage specificity that is balanced with that of the beta-globin gene cluster. In this article, we summarize the research on the control of expression of the alpha-globin gene cluster, mainly with respect to the alpha-major regulatory element (alpha-MRE): HS-40, the tissue-specific and developmental control of its expression, and its chromosomal environment. In summary, the alpha-globin gene cluster is expressed in an open chromosomal environment; HS-40, the 5'-flanking sequence, the transcribed region, and the 3'-flanking sequence interact to fully regulate its expression.

Gene Expression Regulation↗
PubMed2002

Optimizing the delivery systems of chimeric RNA.DNA oligonucleotides.

Special oligonucleotides for targeted gene correction have attracted increasing attention recently, one of which is the chimeric RNA.DNA oligonucleotide (RDO) system. RDOs for targeted gene correction were first designed in 1996, and are typically 68 nucleotides in length including continuous RNA and DNA sequences (RNA is 2'-O-methyl-modified). They have a 25 bp double stranded region homologous to the targeted gene, two hairpin ends of T loop and a 5 bp GC clamp, that give the molecule much greater stability [Fig. 1]. One mismatch site in the middle of the double-stranded region is designed for targeted gene therapy. RDOs have been used recently for targeted gene correction of point mutations both in vitro and in vivo, but many problems must be solved before clinical application. One of the solutions is to optimize the delivery vectors for RDOs. To date, few RDO delivery systems have been used. Therefore, new vectors should be tried for RDO transfer, such as the use of nanoparticles. Additionally, different kinds of modifications should be applied to RDO carrier systems to increase the total correction efficiency in vivo. Only with the development of delivery systems can RDOs be used for gene therapy, and successfully applied to functional genomics.

Chimera↗
PubMed2002

Screening regulatory sequences from bacterial artificial chromosome DNA of alpha- and beta-globin gene clusters.

In the forthcoming postgenomic era, identification of regulatory DNA sequences is becoming increasingly important for characterizing DNA-binding proteins and for elucidating the regulatory mechanisms of gene expression. Presently, there lack efficient methods to broadly screen and identify DNA regulatory elements on a large scale. We established herein an efficient strategy to screen regulatory sequences from bacterial artificial chromosome (BAC) DNAs containing human alpha- and beta-globin gene clusters based on polymerase chain reaction and electrophoretic mobility shift assay (EMSA) techniques without purified transcription factors. Twenty-three subclones derived from alpha-BAC DNA by bulk EMSA selection retained the ability to bind nuclear proteins of K562 cells when retested by EMSA. In 19 clones sequenced, 14 are identical to those registered in GenBank and five have one base difference. All of the 24 randomly picked beta-BAC clones showed specific binding with nuclear proteins of K562 cells. In 11 clones sequenced, eight are identical to those registered in GenBank and three have one base difference. This approach could be particularly powerful if combined with other systematic methods for identifying cis-regulatory DNA elements.

Binding Sites↗
PubMed2002

High-mobility group protein 2 may be involved in the locus control region regulation of the beta-globin gene cluster.

Expression regulation of the beta-globin gene cluster is a result of synergistic interactions between cis-elements and trans-acting factors. Previous studies usually concentrated on the core sequence of each hypersensitive site in the locus control region of the beta-globin gene cluster. But more and more evidence illustrates that the flanking regions are indispensable also. Using electrophoretic mobility shift assay and solid-phase DNase I footprinting methods, we identified a small nuclear protein from K562 cells that binds specifically to the first AT-rich region flanking the hypersensitive site 2 core sequence of the human beta-globin gene locus control region. N-terminal sequencing of the enriched protein proved that it is a member of the high-mobility group protein 2 family. This indicates that the AT-rich region in human hypersensitive site 2 may take part in the regulation of the beta-globin gene cluster by facilitating DNA bending, which is a prerequisite for the looping mechanism in this region.

Base Sequence↗
PubMed2002

[The possible mechanisms underlying low expression of human beta-globin gene cloned in a retroviral vector].

Murine erythroleukemia (MEL) cell line was used as a model to evaluate the potential value of retroviral construct containing human beta-globin express io n cassette in gene therapy of beta-thalassemia and to explore possible mechanisms underlying low expression of retrovirally cloned human beta-globin gene. A recombinant retroviral vector was constructed, which harbored 2.0 kb beta-globin gene w it h a 374 bp deletion in intervening sequence II coupled with a mini locus control region (miniLCR) composed of DNaseI hypersensitive sites (HS) 2 and 3 from human beta-LCR. The recombinant retroviruses were generated from an established psi-2 producer cell line, and by transient transfection of amphotropic packaging cell l ine psi-A, respectively. The integrity of provirus in transduced MEL cells was determined using Southern blot, and the expression of transferred human beta-globin gene was detected using RNase protection assay. The structure of provirus was further analyzed by sequence analysis of PCR products from genomic DNA of MEL individual clone as template. The results demonstrated that the average expression of human beta-globin gene was (52.4-/+11.2)% (n=12) and (73.8-/+14.3)% (n=12, without copy-number determination), compared with that of endogenous murine alpha-globin ge ne, in MEL cells transduced with the recombinant retrovirus from transient transfection of psi-A and MEL cells transfected with the construct, respectively. In M EL cells transduced with virus from psi-2 producer cell line, however, the average expression was less than 3%. A point mutation was detected in HS2 of provirus i n MEL cell clone with low expression of human beta-globin gene. The possible mechanisms involved in low expression, including position effect, DNA methylation a nd RNA interference are discussed in addition to the point mutation.

Base Sequence↗
PubMed1999

Progress in Targeted Gene Integration.

The ultimate goal of gene therapy is correction of the genetic disorders by introducing the genetic materials into targeted cells, and gene expression should be permanent and at an appropriate level. The widely used gene therapy vectors nowadays can not meet the demands. The targeted integration mediated by the homologous recombination is the best strategy for human gene therapy, because the gene expression is controlled by the natural elements. The progress in homologous recombination recently made it possible to correct the genetic disorder s by targeted integration of the interest gene.

Journal Article↗
PubMedDate not supplied

Analysis of adeno-associated virus-mediated ex vivo transferred human beta-globin gene in bone marrow engrafted mice.

Adeno-associated virus (AAV)-2 was developed as a useful vector for human gene therapy. In this report, we analyzed the integration and expression of AAV-mediated ex vivo transferred human beta-globin gene in bone marrow (BM) reconstituted mice. Recombinant AAV (rAAV) containing human beta-globin gene was packaged by infecting individual G418-resistant BHK-21 cell clones integrated with the plasmid AV53HS432Deltabeta2.0Neo with recombinant herpes simplex virus, which can express rep and cap genes of wild-type AAV. The titer of rAAV was determined using slot blot hybridization with a result of 10(13) virus particles/ml (genome copy number). Low-density mononuclear cells were isolated from fetal livers of embryos from pregnant C57BL/6 mice at 14-16 days of gestation and were infected with rAAV. The transduced hematopoietic cells were then reinfused into lethally irradiated C57BL/6 recipient mice via tail vein injection. To analyze the provirus in short-term and long-term BM reconstituted mice, PCR/Southern blot and RT-PCR were performed to identify the integrity of the provirus and to detect the expression of human beta-globin gene, respectively. Genomic DNA was extracted from spleen nodules of BM reconstituted mice 12 days after transplantation. Human beta-globin gene was detected in 1 out of 6 nodules using PCR combined with Southern blot. Human beta-globin gene was also detected in the BM and thymus of mouse Y6161, in the thymus and spleen of mouse Y6162 and in the BM of mice Y6211 and Y6212. RT-PCR revealed low levels of expression of human beta-globin gene in the BM of mouse Y6211. Our results suggested that the efficiency of AAV-mediated human beta-globin gene integration into hematopoietic stem/progenitor cells was very low. It is necessary to perform further research on AAV biology before applying gene therapy that requires integration of a foreign gene into host chromosomes.

Animals↗