Efficient generation of transgenic pigs using equine infectious anaemia virus (EIAV) derived vector.

Traditional methods of transgene delivery in livestock are inefficient. Recently, human immunodeficiency virus (HIV-1) based lentiviral vectors have been shown to offer an efficient transgene delivery system. We now extend this method by demonstrating efficient generation of transgenic pigs using an equine infectious anaemia virus derived vector.

Efficient production of germline transgenic chickens using lentiviral vectors.

An effective method for genetic modification of chickens has yet to be developed. An efficient technology, enabling production of transgenic birds at high frequency and with reliable expression of transgenes, will have many applications, both in basic research and in biotechnology. We investigated the efficiency with which lentiviral vectors could transduce the chicken germ line and examined the expression of introduced reporter transgenes.

Highly efficient EIAV-mediated in utero gene transfer and expression in the major muscle groups affected by Duchenne muscular dystrophy.

Gene therapy for Duchenne muscular dystrophy has so far not been successful because of the difficulty in achieving efficient and permanent gene transfer to the large number of affected muscles and the development of immune reactions against vector and transgenic protein. In addition, the prenatal onset of disease complicates postnatal gene therapy. We have therefore proposed a fetal approach to overcome these barriers.

Long-term transgene expression by administration of a lentivirus-based vector to the fetal circulation of immuno-competent mice.

Inefficient gene transfer, inaccessibility of stem cell compartments, transient gene expression, and adverse immune and inflammatory reactions to vector and transgenic protein are major barriers to successful in vivo application of gene therapy for most genetic diseases. Prenatal gene therapy with integrating vectors may overcome these problems and prevent early irreparable organ damage. To this end, high-dose attenuated VSV-G pseudotyped equine infectious anaemia virus (EIAV) encoding beta-galactosidase under the CMV promoter was injected into the fetal circulation of immuno-competent MF1 mice.

Rabies virus glycoprotein pseudotyping of lentiviral vectors enables retrograde axonal transport and access to the nervous system after peripheral delivery.

In this report it is demonstrated for the first time that rabies-G envelope of the rabies virus is sufficient to confer retrograde axonal transport to a heterologous virus/vector. After delivery of rabies-G pseudotyped equine infectious anaemia virus (EIAV) based vectors encoding a marker gene to the rat striatum, neurons in regions distal from but projecting to the injection site, such as the dopaminergic neurons of the substantia nigra pars compacta, become transduced. This retrograde transport to appropriate distal neurons was also demonstrated after delivery to substantia nigra, hippocampus and spinal cord and did not occur when vesicular stomatitis virus glycoprotein (VSV-G) pseudotyped vectors were delivered to these sites.

Evidence for the role of His-142 of protein 1C in the acid-induced disassembly of foot-and-mouth disease virus capsids.

Foot-and-mouth disease virus (FMDV) capsids are inherently labile under mildly acidic conditions, dissociating to pentamers at pH values in the region of 6.5, with the release of protein 1A and the viral RNA. This acid-induced disassembly is thought to be required for the entry of the virus genome into the host cell.

The structure and function of a foot-and-mouth disease virus-oligosaccharide receptor complex.

Heparan sulfate has an important role in cell entry by foot-and-mouth disease virus (FMDV). We find that subtype O1 FMDV binds this glycosaminoglycan with a high affinity by immobilizing a specific highly abundant motif of sulfated sugars. The binding site is a shallow depression on the virion surface, located at the junction of the three major capsid proteins, VP1, VP2 and VP3.

Arginine-glycine-aspartic acid-specific binding by foot-and-mouth disease viruses to the purified integrin alpha(v)beta3 in vitro.

The integrin alpha(v)beta3 has been shown to act as the receptor for internalization of foot-and-mouth disease virus (FMDV) (A12), with attachment being through a highly conserved RGD motif located on the G-H loop of viral capsid protein VP1. In addition, however, we have recently shown that efficient infection of culture-grown cells by FMDV (O1BFS) requires binding to cell surface heparan sulfate. In this study, we have used a solid-phase receptor binding assay to characterize the binding by FMDV to purified alpha(v)beta3 in the absence of heparan sulfate and other cell surface components.

Efficient infection of cells in culture by type O foot-and-mouth disease virus requires binding to cell surface heparan sulfate.

Foot-and-mouth disease virus (FMDV) enters cells by attaching to cellular receptor molecules of the integrin family, one of which has been identified as the RGD-binding integrin alpha(v)beta3. Here we report that, in addition to an integrin binding site, type O strains of FMDV share with natural ligands of alpha(v)beta3 (i.e.

Bacteriophage lambda-mediated transposon mutagenesis of phytopathogenic and epiphytic Erwinia species is strain dependent.

Using transformation and conjugal mobilization, plasmids carrying the lamB gene of Escherichia coli were transferred to a range of Erwinia strains. The resultant strains were infected with lambda 467, and kanamycin resistant transductants were screened for various mutant phenotypes including auxotrophy and altered extracellular enzyme activities. Reversion analysis suggested that most mutant phenotypes were due to Tn5 insertion.