Efficient transformation system for Propionibacterium freudenreichii based on a novel vector

A 3.6-kb endogenous plasmid was isolated from a Propionibacterium freudenreichii strain and sequenced completely. Based on homologies with plasmids from other bacteria, notably a plasmid from Mycobacterium, a region harboring putative replicative functions was defined. Outside this region two restriction enzyme recognition sites were used for...

Live bacterial delivery systems for development of mucosal vaccines

By expression of foreign antigens in attenuated strains derived from bacterial pathogens and in non-pathogenic commensal bacteria, recombinant vaccines are being developed that aim to stimulate mucosal immunity. Recent advances in the pathogenesis and molecular biology of these bacteria have allowed rational development of new and improved...

Recombinant adenoviral vectors have adjuvant activity and stimulate T cell responses against tumor cells

The host-immune response against adenoviruses forms a major obstacle for their use as gene therapy vectors for treatment of genetic defects. None the less, they are the preferred vectors for in vivo gene transfer in experimental gene therapy protocols for cancer. In this article we demonstrate the antitumor efficacy of adenovirus-mediated...

Expression of the protective antigen of bacillus anthracis by lactobacillus casei: Towards the development of an oral vaccine against anthrax

Bacillus anthracis is the causative organism of the disease anthrax. The ability of the organism to form resistant spores and infect via the aerosol route has led to it being considered as a potential biological warfare agent. The current available human vaccines are far from ideal, they are expensive to produce, require repeated doses and may...

Instruments for oral disease-intervention strategies: Recombinant Lactobacillus casei expressing tetanus toxin fragment C for vaccination or myelin proteins for oral tolerance induction in multiple sclerosis

Lactobacillus strains possess properties that make them attractive candidates as vehicles for oral administration of therapeutics. In this report we describe the construction and analysis of recombinant Lactobacillus casei applicable in oral vaccination against an infectious disease (tetanus) and in oral tolerance induction for intervention in...

Reversal of hyperlipidaemia in apolipoprotein C1 transgenic mice by adenovirus-mediated gene delivery of the low-density-lipoprotein receptor, but not by the very-low-density-lipoprotein receptor

We have shown previously that human apolipoprotein (apo)Cl transgenic mice exhibit hyperlipidaemia, due primarily to an impaired clearance of very-low-density lipoprotein (VLDL) particles from the circulation. In the absence of at least the low-density-lipoprotein receptor (LDLR), it was shown that APOCI overexpression in transgenic mice...

Expression of cytochromes P450 in mammalian cells.

Adenovirus mediated overexpression of human phospholipid transfer protein alters plasma HDL levels in mice

To study the function of plasma phospholipid transfer protein (PLTP) in vivo, a liver directed adenoviral gene transfer system was used to overexpress human PLTP in mice. For the experiments, two strains of mice, wild type (C57/B1) and mice transgenic for the human apoA-I gene (HuApoA- ITg), were utilized. Five days after injection of the...

A convenient and reproducible method to genetically transform bacteria of the genus Bifidobacterium

A protocol was developed for the introduction of foreign plasmid DNA into various Bifidobacterium strains. The method, which is applicable to all Bifidobacterium species tested so far, is based on electroporation of bacteria made competent by preincubation in electroporation buffer for several hours at 4°C. Transformation of Bifidobacterium...

An expression system based on the promoter region of the Aspergillus awamori 1,4-bèta-endoxylanase A gene

A new, highly inducible fungal promoter derived from the Aspergillus awamori 1,4-β-endoxylanase A (exlA) gene is described. Induction analysis, carried out with the wild-type strain in shake flasks, showed that exlA expression is regulated at the transcriptional level. Using a β-glucuronidase (uidA) reporter strategy, D-xylose was shown to be an...

A novel strategy for the isolation of defined pyrG mutants and the development of a site-specific integration system for Aspergillus awamori

A homologous gene transfer system for Aspergillus awamori for site-specific integration is described, based on two components. First, a defined A. awarnori pyrG mutant strain constructed by a selection strategy for gene-replacement in fungi. Second, a vector with a homologous pyrG selection marker containing a defined mutation at a site...

Construction and heterologous expression of a synthetic copy of the cutinase cDNA from Fusarium solani pisi

A copy of the cutinase cDNA from Fusarium solani pisi was constructed starting from synthetic oligonucleotides. For this construction three separate cassettes were made, which were subsequently assembled to form the cutinase gene. Heterologous expression of the synthetic cutinase gene and the subsequent secretion of the recombinant enzyme was...

The effect of multiple copies of the upstream region of the Aspergillus niger glucoamylase-encoding gene on expression

The regulation of transcription of the glucoamylase-encoding gene (glaA) of Aspergillus niger was studied. To facilitate this study a reporter strain containing a fusion of the glaA promoter (P(glaA)) of A. niger to the β- glucuronidase-encoding gene (uidA) of Escherichia coli was constructed. To analyze whether regulatory proteins are involved...

Factors affecting the transduction of pluripotent hematopoietic stem cells: Long-term expression of a human adenosine deaminase gene in mice

An amphotropic retroviral vector, LgAL(ΔMo + PyF101) containing a human adenosine deaminase (ADA) cDNA was used to optimize procedures for the lasting genetic modification of the hematopoietic system of mice. The highest number of retrovirally infected cells in the hematopoietic tissues of long- term reconstituted mice was observed after...

Expression of the Autographa californica nuclear polyhedrosis virus p10 gene: Effect of polyhedrin gene expression

Two major late proteins, polyhedrin and p10, are synthesized in large quantities in baculovirus infected insect cells. This and the fact that both proteins are dispensable for virus replication, form the basis for the use of these viruses as vector for foreign gene expression. To address the question whether the Autographa californica nuclear...

An upstream activating sequence from the Aspergillus nidulans gpdA gene

Introduction of a previously identified promoter element of the Aspergillus nidulans gpdA gene (encoding glyceraldehyde-3-phosphate dehydrogenase), the so-called gpd box, into the upstream region of the highly regulated A. nidulans amdS gene (encoding acetamidase), significantly increased (up to 30-fold) the expression of the lacZ reporter gene...

Intracellular and extracellular production of proteins in Aspergillus under the control of expression signals of the highly expressed Aspergillus nidulans gpdA gene

The expression in Aspergillus is described of genes, coding for intracellular and extracellular proteins controlled by the promoter region of the constitutively and efficiently expressed glyceraldehyde-3-phosphate dehydrogenase gene (gpdA) of Aspergillus nidulans. Both the homologous gpdA and the heterologous Escherichia coli β-galactosidase ...

Expression of human adenosine deaminase in mice transplanted with hemopoietic stem cells infected with amphotropic retroviruses

Amphotropic recombinant retroviruses were generated carrying sequences encoding human adenosine deaminase (ADA). Transcription of the human ADA gene was under control of a hybrid long terminal repeat in which the enhancer from the Moloney murine leukemia virus was replaced by an enhancer from the F101 host-range mutant of polyoma virus....