Multicenter study of the molecular basis of thalassemia intermedia in different ethnic populations.

We studied 325 thalassemia intermedia patients from Iran, India, Pakistan, Thailand, Mauritius and Cyprus to examine factors which influence the phenotype. The beta-thalassemia (thal) mutations were determined for 219 beta-thal/beta-thal and 106 beta-thal/Hb E [beta26(B8)Glu-->Lys, GAG-->AAG] thalassemia intermedia patients. Thirty-one different mutations were identified, and their combination gave rise to more than 44 different genotypes, of which 14 (31.

Method for efficient transfection of in vitro-transcribed mRNA into SK-N-AS and HEK293 cells: difference in the toxicity of nuclear EGFP compared to cytoplasmic EGFP.

Here we report a method for efficient transfection of in vitro-transcribed mRNA into two different types of human adherent cells, the neuroblastoma cell line SK-N-AS, and the transformed kidney cell line HEK293. By using newly trypsinized adherent cells in suspension and Lipofectaminetrade mark 2000, we detected a transfection efficiency of 80-90% in both cell lines and a cell viability of 90% in SK-N-AS and 60% in HEK293, 24 h after transfection when using cytoplasmic enhanced green fluorescent protein (EGFP)-mRNA. We have evaluated the different effects of the generally used EGFP that mainly localizes to the cytoplasm and nuclear EGFP, where the nuclear EGFP are more toxic to the cells than the cytoplasmic EGFP.

A humanized BAC transgenic/knockout mouse model for HbE/beta-thalassemia.

Hemoglobin E (HbE) is caused by a G-->A mutation at codon 26 of the beta-globin gene, which substitutes Glu-->Lys. This mutation gives rise to functional but unstable hemoglobin and activates a cryptic splice site causing mild anemia. HbE reaches a carrier frequency of 60-80% in some Southeast Asian populations.

Humanized beta-thalassemia mouse model containing the common IVSI-110 splicing mutation.

Splicing mutations are common causes of beta-thalassemia. Some splicing mutations permit normal splicing as well as aberrant splicing, which can give a reduced level of normal beta-globin synthesis causing mild disease (thalassemia intermedia). For other mutations, normal splicing is reduced to low levels, and patients are transfusion-dependent when homozygous for the disease.

Introduction of in vitro transcribed ENO1 mRNA into neuroblastoma cells induces cell death.

Background: Neuroblastoma is a solid tumour of childhood often with an unfavourable outcome. One common genetic feature in aggressive tumours is 1p-deletion. The alpha-enolase (ENO1) gene is located in chromosome region 1p36.

A humanized mouse model for a common beta0-thalassemia mutation.

Accurate animal models that recapitulate the phenotype and genotype of patients with beta-thalassemia would enable the development of a range of possible therapeutic approaches. Here we report the generation of a mouse model carrying the codons 41-42 (-TTCT) beta-thalassemia mutation in the intact human beta-globin locus. This mutation accounts for approximately 40% of beta-thalassemia mutations in southern China and Thailand.

The potential of bone marrow stem cells to correct liver dysfunction in a mouse model of Wilson's disease.

Metabolic liver diseases are excellent targets for correction using novel stem cell, hepatocyte, and gene therapies. In this study, the use of bone marrow stem cell transplantation to correct liver disease in the toxic milk (tx) mouse, a murine model for Wilson's disease, was evaluated. Preconditioning with sublethal irradiation, dietary copper loading, and the influence of cell transplantation sites were assessed.

Complementation of alpha-thalassaemia in alpha-globin knockout mice with a 191 kb transgene containing the human alpha-globin locus.

alpha-thalassaemia is an inherited blood disorder caused by a decrease in the synthesis of alpha-globin due to mutations in one or both of the alpha-globin genes located on human chromosome 16. A 191 kb transgene derived from a sequenced bacterial artificial chromosome (BAC) clone carrying the human alpha-globin gene cluster, together with about 100 kb of sequence upstream of DNase1 hypersensitive site HS-40 and 30 kb downstream of the alpha1-globin gene, was introduced into fertilised mouse oocytes by pronuclear microinjection. Three transgenic founder mice were obtained.

Analysis of intergenic transcription in the human IL-4/IL-13 gene cluster.

During the differentiation of naïve CD4+ precursors to T helper 1 (Th1) or Th2 effector cells, several epigenetic changes occur in a lineage-specific manner at the IFN-gamma or IL-4/IL-13 loci. These changes result in alterations in the chromatin structure of these loci and, hence, lineage-restricted expression of the corresponding cytokines. Intergenic transcripts have recently been shown to regulate the expression of genes in the beta-globin locus; therefore, we have examined the Th2 cytokine gene cluster during human Th1/Th2 differentiation and in a transgenic mouse line containing the human IL-4/IL-13 genes for intergenic transcripts.

A knock-out mouse model for methylmalonic aciduria resulting in neonatal lethality.

Methylmalonic aciduria is a human autosomal recessive disorder of organic acid metabolism resulting from a functional defect in the activity of the enzyme methylmalonyl-CoA mutase. Based upon the homology of the human mutase locus with the mouse locus, we have chosen to disrupt the mouse mutase locus within the critical CoA binding domain using gene-targeting techniques to create a mouse model of methylmalonic aciduria. The phenotype of homozygous knock-out mice (mut-/-) is one of early neonatal lethality.

Transgenes encompassing dual-promoter CpG islands from the human TBP and HNRPA2B1 loci are resistant to heterochromatin-mediated silencing.

The genetic elements that are responsible for establishing a transcriptionally competent, open chromatin structure at a region of the genome that consists only of ubiquitously expressed, housekeeping genes are currently unknown. We demonstrate for the first time through functional analysis in stably transfected tissue culture cells that transgenes containing methylation-free CpG islands spanning the dual divergently transcribed promoters from the human TATA binding protein (TBP)-proteasome component-B1 (PSMB1) and heterogeneous nuclear ribonucleoprotein A2/B1 (HNRPA2B1)-heterochromatin protein 1Hs-gamma (chromobox homolog 3, CBX3) gene loci are sufficient to prevent transcriptional silencing and a variegated expression pattern when integrated within centromeric heterochromatin. In addition, only transgene constructs extending over both the HNRPA2B1 and the CBX3 promoters, and not the HNRPA2B1 promoter alone, were able to confer high and stable long-term EGFP reporter gene expression.

Targeted modification of a human beta-globin locus BAC clone using GET Recombination and an I-Scei counterselection cassette.

There is a need for better approaches to allow precise engineering of large genomic BAC DNA fragments, to facilitate the use of intact genomic loci for therapeutic and biotechnology applications. We report an efficient method to insert any modification in any genomic locus, using a human beta-globin locus BAC clone as a model system. The modifications can range from single base changes to large insertions or deletions and leave no operational sequences.

Insertion of modifications in the beta-globin locus using GET recombination with single-stranded oligonucleotides and denatured PCR fragments.

We describe the use of the GET recombination system with oligonucleotides or single-stranded polymerase chain reaction (PCR) fragments to insert modifications in the human beta-globin locus without counterselection. The method involves recombination between oligonucleotides or denatured PCR fragments and homologous sequences in the beta-globin gene in a clone of 205-kb bacterial artificial chromosome (BAC), based on the inducible expression of the recE, recT, and gam genes. In this method, oligonucleotides or denatured PCR fragments are electroporated directly into cells carrying both the globin BAC and the pGETrec plasmid, after induction of the GET recombination system.

Molecular studies in mutase-deficient (MUT) methylmalonic aciduria: identification of five novel mutations.

Mutase-deficient (MUT) methylmalonic aciduria (MMA) is an autosomal recessive inborn error of organic acid metabolism, resulting from a functional defect in the nuclear encoded mitochondrial enzyme methylmalonyl-CoA mutase (MCM) (EC.5.4.

Transcriptional regulation of the human TATA binding protein gene.

The human TATA binding protein (TBP) locus consists of a functional domain of three closely linkedhousekeeping genes (TBP, PSMB1 (proteasomal C5 subunit), and PDCD2 (programmed cell death-2)) within a 50-kb interval at chromosome position 6q27. Here we demonstrate that a genomic clone spanning the 20-kb TBP gene, with 12 kb 5' and 3' flanking sequences, was fully functional in stable, transfected L-cells harboring a single copy of this transgene, including after long-term (60 day) culture in the absence of drug selective pressure. Furthermore, we were only able to detect DNaseI hypersensitive sites at the TBP and PSMB1 promoters present within this 44-kb fragment.