Development and functional testing of a novel in vitro delayed scratch closure assay.

As the development of chronic wound therapeutics continues to expand, the demand for advanced assay systems mimicking the inflammatory wound microenvironment in vivo increases. Currently, this is performed in animal models or in in vitro cell-based models such as cell culture scratch assays that more closely resemble acute wounds. Here, we describe for the first time a delayed scratch closure model that mimics some features of a chronic wound in vitro.

A versatile genomic transgenesis platform with enhanced integrase for human Expi293F cells.

Reliable cell-based platforms to test and/or produce biologics in a sustainable manner are important for the biotech industry. Utilizing enhanced λ integrase, a sequence-specific DNA recombinase, we developed a novel transgenesis platform involving a fully characterized single genomic locus as an artificial landing pad for transgene insertion in human Expi293F cells. Importantly, transgene instability and variation in expression were not observed in the absence of selection pressure, thus enabling reliable long-term biotherapeutics testing or production.

Biomedical applications of glycosylphosphatidylinositol-anchored proteins.

Glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) use a unique posttranslational modification to link proteins to lipid bilayer membranes. The anchoring structure consists of both a lipid and carbohydrate portion and is highly conserved in eukaryotic organisms regarding its basic characteristics, yet highly variable in its molecular details. The strong membrane targeting property has made the anchors an interesting tool for biotechnological modification of lipid membrane-covered entities from cells through extracellular vesicles to enveloped virus particles.

Postexit surface engineering of retroviral/lentiviral vectors.

Gene delivery vectors based on retroviral or lentiviral particles are considered powerful tools for biomedicine and biotechnology applications. Such vectors require modification at the genomic level in the form of rearrangements to allow introduction of desired genes and regulatory elements (genotypic modification) as well as engineering of the physical virus particle (phenotypic modification) in order to mediate efficient and safe delivery of the genetic information to the target cell nucleus. Phenotypic modifications are typically introduced at the genomic level through genetic manipulation of the virus producing cells.

Fluorescence molecular painting of enveloped viruses.

In this study, we describe a versatile, flexible, and quick method to label different families of enveloped viruses with glycosylphosphatidylinositol-modified green fluorescent protein, termed fluorescence molecular painting (FMP). As an example for a potential application, we investigated virus attachment by means of flow cytometry to determine if viral binding behavior may be analyzed after FMP of enveloped viruses. Virus attachment was inhibited by using either dextran sulfate or by blocking attachment sites with virus pre-treatment.

Magnetic field-controlled gene expression in encapsulated cells.

Cell and gene therapies have an enormous range of potential applications, but as for most other therapies, dosing is a critical issue, which makes regulated gene expression a prerequisite for advanced strategies. Several inducible expression systems have been established, which mainly rely on small molecules as inducers, such as hormones or antibiotics. The application of these inducers is difficult to control and the effects on gene regulation are slow.

The 5'-untranslated region of the mouse mammary tumor virus mRNA exhibits cap-independent translation initiation.

In this study, we demonstrate the identification of an internal ribosome entry site (IRES) within the 5'-untranslated region (5'-UTR) of the mouse mammary tumor virus (MMTV). The 5'-UTR of the full-length mRNA derived from the infectious, complete MMTV genome was cloned into a dual luciferase reporter construct containing an upstream Renilla luciferase gene (RLuc) and a downstream firefly luciferase gene (FLuc). In rabbit reticulocyte lysate, the MMTV 5'-UTR was capable of driving translation of the second cistron.

Rev regulates translation of human immunodeficiency virus type 1 RNAs.

Full-length human immunodeficiency virus type 1 (HIV-1) RNA acts as both mRNA, encoding Gag and Gag-Pol polyproteins, and genomic RNA. Translation of this RNA must be tightly controlled to allow sufficient protein synthesis prior to a switch to particle production. The viral protein Rev stimulates nuclear export of unspliced HIV-1 RNAs containing the Rev response element, but may also stimulate translation of these RNAs.

Rafts, anchors and viruses--a role for glycosylphosphatidylinositol anchored proteins in the modification of enveloped viruses and viral vectors.

Lipid rafts have been proposed as sites for the assembly of a number of viruses and are considered to play a major role in pseudotyping events. As a consequence, host glycosylphosphatidylinositol (GPI) anchored proteins commonly associated with lipid rafts can be found being incorporated into viral lipid envelopes with beneficial consequences for viral replication. In this review we will look at the link between lipid rafts, GPI-anchored proteins and retroviral particles and how these relationships can be exploited for the modification of enveloped viruses.

Association of glycosylphosphatidylinositol-anchored protein with retroviral particles.

We describe for the first time the association of glycosylphosphatidylinositol (GPI) -anchored proteins with retroviral and lentiviral particles, similar to a process well established for cells, termed "painting." The aim of the study was to assess the feasibility of modification of retroviral vectors by exogenous addition of recombinant protein, removing the need for genetic engineering of virus producer cell lines. The recombinant GPI protein CD59his was purified via fast protein liquid chromatography and associated with concentrated virus stock in a controlled incubation procedure.

Mutation of the Rev-binding loop in the human immunodeficiency virus 1 leader causes a replication defect characterized by altered RNA trafficking and packaging.

An internal RNA loop, located within the packaging signal of human immunodeficiency virus 1, that resembles the Rev-responsive element (RRE) closely was identified previously. Subsequent in vitro studies confirmed that the loop, termed loop A, could bind Rev protein specifically. Its proximity to the major splice donor has suggested a role for Rev-loop A interaction supplementary to or preceding that of the Rev-RRE interaction.

Enhancement of the StreptoTag method for isolation of endogenously expressed proteins with complex RNA binding targets.

StreptoTag is a novel affinity chromatography-based method for the isolation of high- and low-affinity RNA binding proteins. Originally it was shown possible to isolate recombinant protein from yeast or bacterial extracts using small, specific, well-characterised RNA binding targets. Here we show that using an enhanced aptamer it is not only possible to efficiently immobilise large, highly structured RNA binding targets onto the streptomycin columns but also that the StreptoTag method can be used for the isolation and purification of endogenously expressed regulatory proteins, with relatively low abundance, from eukaryotic extracts.

MMTV accessory factor Naf affects cellular gene expression.

Mouse mammary tumour virus (MMTV) encodes a viral superantigen (Sag) and a negative acting factor (Naf) which share parts of their coding sequence. Using 2-dimensional gel electrophoresis (2D-DIGE), we could show that at least 10 different cellular proteins were differentially expressed in Naf positive cells. Also, luciferase reporter expression was down-regulated in Naf expressing cells independent of the promoter used and further experiments suggested that this effect was due in part to a decrease in cellular growth rates.

HIV-1 Rev can specifically interact with MMTV RNA and upregulate gene expression.

We present evidence that the HIV-1 Rev protein can heterologously regulate expression of the simple beta retrovirus mouse mammary tumour virus (MMTV). Up to 10-fold upregulation was seen in a functional assay system when specific MMTV sequences were substituted for the HIV-1 Rev responsive element (RRE). RNA gel shift analysis showed that purified recombinant Rev could specifically bind to MMTV unique region 3 prime (U3) RNA and that these sequences could compete for wild-type Rev-RRE binding approximately 20-fold more efficiently than a non-specific competitor RNA.