Elimination of contaminant Escherichia coli chromosomal DNA from preparations of P1 artificial chromosome recombinants facilitates directed subcloning.

The subcloning of large inserts (>50 kbp) from P1-derived artificial chromosomes (PACs) was found to be hindered by the presence of contaminating Escherichia coli chromosomal fragments which, because of their smaller median size, are recovered preferentially as unwanted subclones. A significant fraction of contaminating DNA was seen to persist after conventional plasmid purification methods. We describe a rigorous protocol for eliminating the bulk of contamination that involves plasmid isolation on commercially available silica-based columns followed by three pulsed field gel electrophoresis steps.

pSURF-2, a modified BAC vector for selective YAC cloning and functional analysis.

A modified bacterial artificial chromosome (BAC) vector, pSURF-2, adapted for the selective subcloning of yeast artificial chromosome (YAC) sequences was constructed. DH10B-U, a pyrF derivative of the highly transformable E. coli strain DH10B was also constructed and used for the detection of Ura+ recombinants carrying DNA linked to YAC right arms.

Rapid quantitation of gene therapy specific CFTR expression using the amplification refractory mutation system.

Gene therapy offers the potential of correcting genetic disorders such as cystic fibrosis (CF). By complementing the non-functional endogenous cystic fibrosis transmembrane conductance regulator (CFTR) gene with a functional transgene, we anticipate it may alleviate the disease phenotype. All approaches to CF gene therapy rely upon sensitive assays to monitor delivery, expression and maintenance of CFTR vectors.

A zinc finger truncation of murine WT1 results in the characteristic urogenital abnormalities of Denys-Drash syndrome.

The Wilms tumor-suppressor gene, WT1, plays a key role in urogenital development, and WT1 dysfunction is implicated in both neoplastic (Wilms tumor, mesothelioma, leukemias, and breast cancer) and nonneoplastic (glomerulosclerosis) disease. The analysis of diseases linked specifically with WT1 mutations, such as Denys-Drash syndrome (DDS), can provide valuable insight concerning the role of WT1 in development and disease. DDS is a rare childhood disease characterized by a nephropathy involving mesangial sclerosis, XY pseudohermaphroditism, and/or Wilms tumor (WT).

Identification and characterization of a homozygous deletion found in ovarian ascites by representational difference analysis.

We have performed representational difference analysis (RDA) on DNA from tumor cells and normal fibroblasts isolated from the ascites of a patient with ovarian cancer. Five of six products of the RDA were homozygously deleted from the tumor DNA. One of these products has been characterized and identifies a homozygous deletion of approximately 6.

A long-range restriction map across 3 Mb of the chromosome 11 breakpoint region of a translocation linked to schizophrenia: localization of the breakpoint and the search for neighbouring genes.

A balanced t(1;11)(q42.1;q14.3) translocation segregates with schizophrenia and related mental illness in a single large Scottish pedigree.

A new yeast artificial chromosome vector designed for gene transfer into mammalian cells.

This report describes the construction of a new yeast artificial chromosome (YAC) vector designed for gene transfer into mammalian cells. For ease of use, the two arms of the vector were cloned separately. The vector harbours the Neo and Hyg genes for dominant selection in mammalian cells, a putative human origin of replication, a synthetic matrix attachment region and two loxP sites (one on each arm).

Gene therapy for cystic fibrosis: a psychosocial study of trial participants.

As yet little is known of psychological aspects of gene therapy. This pilot study aimed to investigate the attitudes, expectations, knowledge and psychological functioning of participants in a phase l safety trial of a single application of gene therapy to cystic fibrosis patients. Sixteen patients were assessed before and after the trial and at a 16 week follow-up using a semi-structured qualitative interview.

Inclusion of cholesterol in DOTAP transfection complexes increases the delivery of DNA to cells in vitro in the presence of serum.

The contrast between the relative efficiency of transfection by cationic lipid reagents in vitro and that in vivo is well recognised. One suggested reason for this is the presence of competing polyanionic surfaces in blood and other biological fluids, and even in vitro transfections have to be performed in low-serum medium. In this study we have shown that by preparing cationic lipid reagents based on DOTAP with cholesterol as a second constituent of the bilayer we can achieve significant levels of in vitro transfection in serum concentrations of up to 80% (DOTAP alone did not transfect at all in these conditions).

Effect of IBMX and alkaline phosphatase inhibitors on Cl- secretion in G551D cystic fibrosis mutant mice.

Some cystic fibrosis transmembrane conductance regulator (CFTR) mutations, such as G551D, result in a correctly localized Cl- channel at the cell apical membrane, albeit with markedly reduced function. Patch-clamp studies have indicated that both phosphatase inhibitors and 3-isobutyl-1-methylxanthine (IBMX) can induce Cl- secretion through the G551D mutant protein. We have now assessed whether these agents can induce Cl- secretion in cftrG551D mutant mice.

Correlation between nasal potential difference measurements, genotype and clinical condition in patients with cystic fibrosis.

In cystic fibrosis (CF), the clinical condition of patients correlates poorly with genotype. One possible explanation is that clinical status is influenced by net preserved chloride secretion rather than the CF mutation. We tested the relationships between residual chloride secretion, as measured by nasal potential difference (PD) and the type of mutation (genotypes expressing apical cystic fibrosis transmembrane conductance regulator (CFTR) protein versus those that do not) and clinical status.

Gene therapy for lung inflammatory diseases: not so far away?

The lung is a readily accessible target organ for gene therapy. To date, therapeutic gene delivery has largely focused on introducing functional, corrective genes in lung diseases arising from single gene defects such as cystic fibrosis. More recently interest has centred on gene therapy as a potential therapeutic tool in modulating complex pathological processes such as pulmonary inflammation.

Evidence for safety and efficacy of DOTAP cationic liposome mediated CFTR gene transfer to the nasal epithelium of patients with cystic fibrosis.

In cystic fibrosis (CF), mutation of the cystic fibrosis transmembrane conductance regulator (CFTR) gene results in defective transepithelial ion transport, leading to life shortening inflammatory lung disease. Before lung studies, we tested the safety and efficacy of gene delivery to the nasal epithelium of CF patients using pCMV-CFTR-DOTAP cationic liposome complex. A single dose of 400 micrograms pCMV-CFTR:2.

Early alterations in airway mucociliary clearance and inflammation of the lamina propria in CF mice.

In cystic fibrosis (CF), whether cystic fibrosis transmembrane conductance regulator (CFTR) dysfunction leads to decreased mucociliary clearance and mucus hypersecretion, before bacterial infection, remains an open question. To answer this question, we quantified in a blind trial the mucociliary transport velocity, the histological state, and the degree of inflammation of the tracheal mucosa in 23 cftr(m1HGU/cftr(m1HGU) transgenic mice (Dorin, J. R.

Novel transcribed sequences neighbouring a translocation breakpoint associated with schizophrenia.

A 1.3Mb chromosome 11-specific yeast artificial chromosome (YAC) that spans a t(1;11) translocation breakpoint associated with major psychosis has been used to enrich cDNAs that are encoded within it and expressed in the human foetal brain. Database analysis of the selected fragments led to the identification of 54 clones matching alpha-tubulin, 4 fragments matching two anonymous human expressed sequence tags (ESTs) and 8 fragments giving no database matches.

An allelic association study of two polymorphic markers in close proximity to a balanced translocation t(1:11) that co-segregates with mental illness.

We report a case control association study using polymorphic markers D1S1621 and D11S931 in unrelated individuals with schizophrenia, unipolar depression and a matched control group. The two polymorphic markers were identified during the positional cloning of the translocation breakpoint t(1:11)(q43:q14.3) that cosegregates with schizophrenia and affective disorders.

Physical mapping of a glutamate receptor gene in relation to a balanced translocation associated with schizophrenia in a large Scottish family.

The metabotropic glutamate receptor subtype 5a (mGluR5a) gene has been localised on the Gene Map of the Human Genome to chromosome 11q, approximately 1 cM from the genetic marker D11S931. D11S931 has been shown to lie close to a translocation breakpoint associated with schizophrenia and other psychiatric disorders in a large Scottish family. Because glutamate receptor genes are excellent candidates for psychiatric disorders, we have investigated the physical distance of this gene from the translocation breakpoint on chromosome 11.

Laboratory and clinical studies in support of cystic fibrosis gene therapy using pCMV-CFTR-DOTAP.

The first phase I study of cystic fibrosis gene therapy using cationic liposomes to deliver the cystic fibrosis conductance regulator gene to the nose reported partial and transient correction of the nasal transepithelial ion transport defect, While encouraging, further improvements will be required if this form of treatment is to be of therapeutic value. We tested a new formulation, pCMV-CFTR-DOTAP. The complex is stable for 10 days and effective at correcting the electrophysiological deficit in the trachea of CF mutant mice at 8 or 9 days after intratracheal instillation.

Plasmid DNA molecules complexed with cationic liposomes are protected from degradation by nucleases and shearing by aerosolisation.

The cationic liposome DOTAP was complexed with plasmid DNA encoding beta-galactosidase in various ratios. As the concentration of DOTAP increased, the DNA became increasingly refractory to staining with ethidium bromide, presumably because the DNA was becoming condensed and being encapsulated by the liposomes. Transfection by DNA-DOTAP complexes at all ratios tested was unaffected by treatment of the complexes with DNase I.

A demonstration using mouse models that successful gene therapy for cystic fibrosis requires only partial gene correction.

Quantifying the level of transgene expression necessary for phenotypic effect is an important consideration in designing somatic gene therapy protocols. A nonlinear relationship between phenotype and gene activity is predicted by control analysis for any autosomal recessive condition. The unaffected phenotype of heterozygotes for autosomal recessive disorders demonstrates that 50% of the normal level of gene expression is sufficient to prevent disease.

Novel transcribed sequences represented in the complex genomic region 5q13.

YACs from the complex repetitive human genomic region 5q13, spanning the spinal muscular atrophy (SMA) locus, have been searched for transcribed sequences using the method of End Ligation Coincident Sequence Cloning. Six transcripts (PT1-6) have been identified, three of which (PT4, PT5 and PT6) are novel. Five of these elements hybridise to multiple loci in 5q13, but PT5 is single copy and maps very close to markers that show linkage disequilibrium with SMA.