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Charcot-Marie-Tooth disease type 4J (CMT4J) is caused by recessive, loss-of-function mutations in FIG4, encoding a phosphoinositol(3,5)P2-phosphatase. CMT4J patients have both neuron loss and demyelination in the peripheral nervous system, with vacuolization indicative of endosome/lysosome trafficking defects. Although the disease is highly variable, the onset is often in childhood and FIG4 mutations can dramatically shorten life span. There is currently no treatment for CMT4J. Here, we present the results of preclinical studies testing a gene-therapy approach to restoring FIG4 expression. A mouse model of CMT4J, the Fig4-pale tremor (plt) allele, was dosed with a single-stranded adeno-associated virus serotype 9 (AAV9) to deliver a codon-optimized human FIG4 sequence. Untreated, [Fig4.sup.plt/plt] mice have a median survival of approximately 5 weeks. When treated with the AAV9-FIG4 vector at P1 or P4, mice survived at least 1 year, with largely normal gross motor performance and little sign of neuropathy by neurophysiological or histopathological evaluation. When mice were treated at P7 or P11, life span was still significantly prolonged and peripheral nerve function was improved, but rescue was less complete. No unanticipated adverse effects were observed. Therefore, AAV9-mediated delivery of FIG4 is a well-tolerated and efficacious strategy in a mouse model of CMT4J., Introduction Charcot-Marie-Tooth disease (CMT) is a collection of inherited motor and sensory neuropathies. Mutations in over 80 genes can lead to CMT, and it is highly heterogeneous (1). Loss of [...]

Adeno-associated virus (AAV) vectors are currently the leading candidates for virus-based gene therapies because of their broad tissue tropism, non-pathogenic nature and low immunogenicity (1). They have been successfully used [...]

Rift Valley fever (RVF) is an acute disease of domestic ruminants in Africa and the Arabian Peninsula. This disease is caused by a mosquito-borne virus of the family Bunyaviridae and [...]

Lymphocytic choriomeningitis virus (LCMV) exhibits natural tropism for dendritic cells and represents the prototypic infection that elicits protective [CD8.sup.+] T cell (cytotoxic T lymphocyte (CTL)) immunity. Here we have harnessed the immunobiology of this arenavirus for vaccine delivery. By using producer cells constitutively synthesizing the viral glycoprotein (GP), it was possible to replace the gene encoding LCMV GP with vaccine antigens to create replication-defective vaccine vectors. These rLCMV vaccines elicited CTL responses that were equivalent to or greater than those elicited by recombinant adenovirus 5 or recombinant vaccinia virus in their magnitude and cytokine profiles, and they exhibited more effective protection in several models. In contrast to recombinant adenovirus 5, rLCMV failed to elicit vector-specific antibody immunity, which facilitated re-administration of the same vector for booster vaccination. In addition, rLCMV elicited T helper type 1 [CD4.sup.+] T cell responses and protective neutralizing antibodies to vaccine antigens. These features, together with low seroprevalence in humans, suggest that rLCMV may show utility as a vaccine platform against infectious diseases and cancer., Vaccines represent one of the most successful interventions in modern medicine, having led to the control of many devastating infectious diseases and recently also to the prevention of cervical cancer. [...]

Typical reverse genetics systems for generating influenza viruses require the insertion of each genome segments by DNA ligation into vectors for genome synthesis and expression. Herein is described the construction and use of a novel pair of plasmid vectors for cloning all eight genome segments of influenza A virus by homologous recombination for influenza virus reverse genetics. Plasmids, pLLBA and pLLBG, were constructed to possess opposing RNA polymerase I and RNA polymerase II transcription units for generating influenza genomic and messenger RNAs, respectively. In addition these promoters flanked a recombination cassette which comprised the conserved 5' (13bp) and 3' (12bp) terminal promoters of influenza virus. These vectors differed due to the presence of an A or a G (plus sense) to correspond to differences at nucleotide position 4 among negative-sense influenza virus promoters. The cloning approach involved homologous recombination of each influenza gene segment and the appropriate linearized pLLBA or pLLBG vectors in E. coli. Direct cloning by recombination was simpler and faster than conventional restriction digestion and ligation methods. This new vector system was successfully used to clone and rescue various influenza viruses and thus has the potential to promote the rapid analysis and vaccine development of novel influenza strains. KEYWORDS: Influenza A virus, reverse genetics, vector construction, homologous recombination, INTRODUCTION Reverse genetics methods have become essential for the genetic manipulation of influenza viral genomes in both basic and applied research (Neumann and Kawaoka, 2001; Subbarao and Katz, 2004; Marsh [...]

Recombinant adeno-associated viruses (AAVs) have been used widely for in vivo gene therapy. However, adaptive immune responses to AAV have posed a significant hurdle in clinical application of AAV vectors. Recent advances have suggested a crucial role for innate immunity in shaping adaptive immune responses. How AAV activates innate immunity, and thereby promotes AAV-targeted adaptive immune responses, remains unknown. Here we show that AAV activates mouse plasmacytoid DCs (pDCs) via TLR9 to produce type I IFNs. In vivo, the TLR9-MyD88 pathway was crucial to the activation of [CD8.sup.+] T cell responses to both the transgene product and the AAV capsid, leading to loss of transgene expression and the generation of transgene product--specific and AAV-neutralizing antibodies. We further demonstrate that TLR9-dependent activation of adaptive immunity targeting AAV was mediated by type I IFNs and that human pDCs could be activated in vitro to induce type I IFN production via TLR9. These results reveal an essential role for the TLR9-MyD88-type I IFN pathway in induction of adaptive immune responses to AAV and suggest that strategies that interfere with this pathway may improve the outcome of AAV-mediated gene therapy in humans., Introduction The adeno-associated virus (AAV) is a non-enveloped, single-stranded DNA virus with a genome of approximately 5 kb. It is a member of the Parvoviridae family and requires a helper [...]

Major limitations to gene therapy using HSCs are low gene transfer efficiency and the inability of most therapeutic genes to confer a selective advantage on the gene-corrected cells. One approach to enrich for gene-modified cells in vivo is to include in the retroviral vector a drug resistance gene, such as the P140K mutant of the DNA repair enzyme [O.sup.6]-methylguanine-DNA methyltransferase (MGMT *). We transplanted 5 rhesus macaques with [CD34.sup.+] cells transduced with lentiviral vectors encoding MGMT * and a fluorescent marker, with or without homeobox B4 (HOXB4), a potent stem cell self-renewal gene. Transgene expression and common integration sites in lymphoid and myeloid lineages several months after transplantation confirmed transduction of long-term repopulating HSCs. However, all animals showed only a transient increase in gene-marked lymphoid and myeloid cells after [O.sup.6]-benzylguanine (BG) and temozolomide (TMZ) administration. In 1 animal, cells transduced with MGMT * lentiviral vectors were protected and expanded after multiple courses of BG/TMZ, providing a substantial increase in the maximum tolerated dose of TMZ. Additional cycles of chemotherapy using 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU) resulted in similar increases in gene marking levels, but caused high levels of nonhematopoietic toxicity. Inclusion of HOXB4 in the MGMT * vectors resulted in no substantial increase in gene marking or HSC amplification after chemotherapy treatment. Our data therefore suggest that lentivirally mediated gene transfer in transplanted HSCs can provide in vivo chemoprotection of progenitor cells, although selection of long-term repopulating HSCs was not seen., Introduction Retroviral vector--mediated gene transfer into HSCs has the potential to improve treatment for various genetic, malignant, and infectious diseases (1), (2). However, a critical limitation to stem cell gene [...]

A variety of viruses establish latency by integrating their genome into the host genome. The integration event generally occurs in a nonspecific manner, precluding the prediction of functional consequences from resulting disruptions of affected host genes. The nonpathogenic adeno-associated virus (AAV) is unique in its ability to stably integrate in a site-specific manner into the human MBS85 gene. To gain a better understanding of the integration mechanism and the consequences of MBS85 disruption, we analyzed the molecular structure of AAV integrants in various latently infected human cell lines. Our study led to the observation that AAV integration causes an extensive but partial duplication of the target gene. Intriguingly, the molecular organization of the integrant leaves the possibility that a functional copy of the disrupted target gene could potentially be preserved despite the resulting rearrangements. A latently infected, Mbs85-targeted mouse ES cell line was generated to study the functional consequences of the observed duplication-based integration mechanism. AAV-modified ES cell lines continued to self-renew, maintained their multilineage differentiation potential and contributed successfully to mouse development when injected into blastocysts. Thus, our study reveals a viral strategy for targeted genome addition with the apparent absence of functional consequences. embryonic stem cells | MBS85 | gene targeting | Rep | AAVS1

[gamma]-Retroviral vectors ([gamma]RVs), which are commonly used in gene therapy, can trigger oncogenesis by insertional mutagenesis. Here, we have dissected the contribution of vector design and viral integration site selection (ISS) to oncogenesis using an in vivo genotoxicity assay based on transplantation of vector-transduced tumor-prone mouse hematopoietic stem/progenitor cells. By swapping genetic elements between [gamma]RV and lentiviral vectors (LVs), we have demonstrated that transcriptionally active long terminal repeats (LTRs) are major determinants of genotoxicity even when reconstituted in LVs and that self-inactivating (SIN) LTRs enhance the safety of [gamma]RVs. By comparing the genotoxicity of vectors with matched active LTRs, we were able to determine that substantially greater LV integration loads are required to approach the same oncogenic risk as [gamma]RVs. This difference in facilitating oncogenesis is likely to be explained by the observed preferential targeting of cancer genes by [gamma]RVs. This integration-site bias was intrinsic to [gamma]RVs, as it was also observed for SIN [gamma]RVs that lacked genotoxicity in our model. Our findings strongly support the use of SIN viral vector platforms and show that ISS can substantially modulate genotoxicity., Introduction Integrative viral vectors commonly used in gene therapy may trigger oncogenesis as a consequence of insertional mutagenesis (1), (2). Because [gamma]-retroviral vector ([gamma]RV) gene transfer into hematopoietic stem/progenitor cells [...]

Gene therapy requires efficient gene delivery to cure or prevent disease by modifying the genome of somatic cells. However, gene vectors, which insert themselves into the host genome in order to achieve persistent protein expression, can trigger oncogenesis by upregulating cellular protooncogenes. This adverse event, known as insertional mutagenesis, has become a major hurdle in the field. Vectors developed on the basis of lentiviruses are considered to be less genotoxic than the hitherto used [gamma]-retroviral vectors. For their report in this issue of the JCI, Montini et al. utilized a tumor-prone mouse model to identify the genetic determinants of insertional mutagenesis (see the related article beginning on page 964). They report that the lentiviral integration pattern and additional improvements in vector design reduce the genotoxic risk. These findings will inform future vector design with the goal of limiting genotoxicity for gene therapy or increasing genotoxicity for protooncogene discovery., The development of safe and efficient methods to modify the genome of somatic cells would provide new perspectives in the prevention, treatment, or even causal cure of inherited and acquired [...]

Transduction of the primate cortex with adeno-associated virus (AAV)-based gene therapy vectors has been challenging, because of the large size of the cortex. We report that a single infusion of AAV2 vector into thalamus results in widespread expression of transgene in the cortex through transduction of widely dispersed thalamocortical projections. This finding has important implications for the treatment of certain genetic and neurodegenerative diseases. adeno-associated viral vector | cortex | gene delivery | thalamocortical

Vectors derived from adeno-associated virus (AAV) are promising for human gene therapy, including treatment for retinal blindness. One major limitation of AAVs as vectors is that AAV cargo capacity has been considered to be restricted to 4.7 kb. Here we demonstrate that vectors with an AAV5 capsid (i.e., rAAV2/5) incorporated up to 8.9 kb of genome more efficiently than 6 other serotypes tested, independent of the efficiency of the rAAV2/5 production process. Efficient packaging of the large murine Abca4 and human MYO7A and CEP290 genes, which are mutated in common blinding diseases, was obtained, suggesting that this packaging efficiency is independent of the specific sequence packaged. Expression of proteins of the appropriate size and function was observed following transduction with rAAV2/5 carrying large genes. Intraocular administration of rAAV2/5 encoding ABCA4 resulted in protein localization to rod outer segments and significant and stable morphological and functional improvement of the retina in Abca4-/- mice. This use of rAAV2/5 may be a promising therapeutic strategy for recessive Stargardt disease, the most common form of inherited macular degeneration. The possibility of packaging large genes in AAV greatly expands the therapeutic potential of this vector system., Introduction Vectors derived from the small, icosahedral, single-stranded DNA adeno-associated virus (AAV) are very promising for gene therapy of human diseases (1). The safety and efficacy of recombinant AAV (rAAV) [...]

Author(s): Thomas R Bauer, Jr [1]; James M Allen [2]; Mehreen Hai [1]; Laura M Tuschong [1]; Iram F Khan [2]; Erik M Olson [2]; Rima L Adler [1]; Tanya [...]

Recombinant adeno-associated virus (rAAV) vectors were used in human trials as carriers of vaccines for HIV-1 after encouraging preclinical results. However, the clinical trials yielded disappointing results. Here we demonstrated that in mice, rAAV vectors expressing the gene encoding HIV-1 gag stimulated gag-specific [CD8.sup.+] T cells, but these T cells failed to expand after a booster immunization with a replication-defective adenoviral (Ad) vector also expressing gag. We tested rAAV vectors of different serotypes expressing HIV-1 gag for induction of transgene product-specific [CD8.sup.+] T cells and found that the immunoinhibitory effect of rAAV priming observed with different AAV serotypes was transgene product specific, was independent of the interval between prime and boost, and extended to boosts with vaccine modalities other than Ad vectors. rAAV vector- induced [CD8.sup.+] T cells proliferated poorly, produced low levels of IFN-[gamma] in response to gag stimulation, and upregulated immunoinhibitory molecules. These T cells did not protect efficiently against challenge with a surrogate pathogen. Finally, we showed that the impaired proliferative capacity of the T cells was caused by persistence of the antigen-encoding rAAV vectors and could be reversed by placing the [CD8.sup.+] T cells in an antigen-free environment. Our data suggest that rAAV vectors induce functionally impaired T cells and could dampen the immune response to a natural infection., Introduction Vectors derived from adeno-associated viruses (AAVs) were initially developed for gene replacement therapy (1-3). They were shown to achieve sustained expression of the therapeutic protein in target tissues of [...]

Byline: Steven W. Warmann (1), Sorin Armeanu (2), Heike Heitmann (1), Peter Ruck (3), Guido Seitz (1), Johannes T. Wessels (4), Marie-Luise Lemken (2), Ulrich M. Lauer (2), Jorg Fuchs (1), Michael Bitzer (2) Keywords: Hepatoblastoma; Viral vectors; Gene therapy; Liver specific promoters Abstract: Gene targeting is currently of distinct interest as an innovative additive treatment option in various malignancies. Its role in pediatric liver tumors has not yet been evaluated thoroughly. For the first time the authors systematically analyzed both lipid-based transfection as well as transduction with adenovirus vectors (Ad) and Sendai virus vectors (SeVV) in order to optimize gene transfer into hepatoblastoma (HB) cells. Two HB cell lines were infected with Ad or SeVV coding for green fluorescent protein (Ad-GFP, SeVV-GFP) transduction efficiencies and apoptosis were assessed using flow cytometry. Furthermore, lipofection of HB cell lines with plasmid-constructs comprising liver-specific promoters was performed using Lipofectamine[TM] 2000 and FuGENE 6[TM] lipofection efficiency was monitored by flow cytometry, microscopy, and luciferase activity. The Ad-GFP showed higher transduction rates (61--86%) than the SeVV-GFP (4--24%) depending on the HB cell line used. Infections with first generation SeVV vectors (SeVV-GFP) led to increased target cell apoptosis (7--43%) compared to Ad-GFP (4--16%). The Lipofectamine[TM] 2000 revealed a higher transfection efficiency than the FuGENE 6[TM] for both HB cell lines tested. The liver-specific promoters were found to be differently active in the HB cell lines. This study delineates recombinant adenovirus vectors as a promising tool for gene transduction in the HB cells. Furthermore, enhanced activity of the liver-specific promoters in HUH6 cells compared to HepT1 cells supports the observation of varying biological behavior in histologically differing HB tissues. Author Affiliation: (1) Department of Pediatric Surgery, University of Tubingen, Hoppe-Seyler-Str.3, 72076, Tubingen, Germany (2) Internal Medicine I, Medical University Clinic Tubingen, Otfried-Muller-Str. 10, 72076, Tubingen, Germany (3) Institute of Pathology, Hospital Leonberg, 71229, Leonberg, Germany (4) Centre Internal Medicine, Department of Nephrology and Rheumatology, Robert-Koch-Str. 40, 37075, Gottingen, Germany Article History: Registration Date: 27/06/2006 Accepted Date: 26/06/2006 Online Date: 29/07/2006 Article note: S. W. Warmann and S. Armeanu shared equally in the authorship of this paper and J. Fuchs and M. Bitzer share the senior authorship.

Results demonstrate that pHT128 plasmid, derived from the broad-host-range vector pGSS33 of Escherichia coli, is transferable into Neisseria meningitidis by triparental conjugation. The plasmid multiplies to the same degreee as in E. coli and stays as an episome. Data point out that pHT128 is useful as a new genetic tool.

Anti-Ras single-chain FV antibody fragments are constructed from secretion vectors of the two yeast species. Results show that both species secrete soluble and functional anti-Ras single-chain Fv antibody fragments and possess the potential for industrial scale production of this recombinant product.

The ability to introduce and express foreign genes can greatly facilitate in vitro studies of the molecular biology of an organism. We report here the first use of modified retroviral vectors to achieve foreign gene expression in primary cultured cells from the Oka organ and ovary of the blue shrimp Penaeus stylirostris (Litopenaeus) stylirostris (Holthuis 1980). Of the heterologous promoters tested, the Moloney murine leukemia virus and Rous sarcoma virus promoters mediated the highest levels of reporter gene expression. Firefly luciferase and Escherichia coli [beta]-galactosidase were successfully used as reporter genes in cultured cells from both Oka organ and ovary. These in vitro systems can be exploited for the study of shrimp cellular biology and regulation of gene expression.

A novel insertion sequence from Mycobacterium fortuitum has been isolated by using a trap vector developed out of inactivation of a lacZ reporter gene. The trap vector seems to be useful in trapping insertion sequences from different mycobacteria by screening for the disrupted LacZ phenotype. One of the predicted proteins had homology to what is said to be a transposase from Acetobacter pasteurianus.

The future of virology is discussed. Gene therapy will be dependent on the development of viral vectors. Recombinant and mutant viruses will be used in vaccines and gene replacement therapy. They will be used for selective killing of tumor cells. The viruses responsible for influenza epidemics might be the most important emerging and evolving one in the future because of demographic factors, with people over the age of 60 making up a greater proportion of the populations of many countries and more people in urban areas.

Research was conducted to determine the action of fosfomycin on MurA enzyme and the effect of amino acid substitution on its activity. Results show that fosfomycin inhibits the enzyme and the inhibitory effect is altered when cysteine residue is replaced with aspartate-117.

The construction of a series of broad-host-range, multipurpose cloning and expression vectors based on the RK2 replicon is described. The expression vectors are synthesized by insertion of the inducible Pu or Pm promoter with its regulatory gene xylR or xylS, respectively, from the TOL plasmid of Pseudomonas putida. In Xanthomonas campestris, expression of the gene encoding the enzyme phosphoglucomutase (CelB) from Acetobacter xylinum shows the inability of the celB gene to complement the role of phosphoglucomutase-phosphomannomutase gene in xylan synthesis.

A recombinant megalovirus promoter was utilized for gene transfer in the pericardial sac of doxycycline-treated hearts to determine the efficiency of the technique as an alternative to intravascular gene transfer. Gene transfer in canine hearts was accomplished by utilizing the human adenovirus serotype-2 recombinant vector which is injected in the pericardial sac. The technique produced extensive transfection of the visceral and parietal pericardium of canine hearts.

Plasmids encoding various external guide sequences (EGSs) were constructed and inserted into Escherichia coli. In strains harboring the appropriate plasmids, the expression of fully induced [beta]-galactosidase and alkaline phosphatase activity was reduced by more than 50%, white no reduction in such activity was observed in strains with nonspecific EGSs. The inhibition of gene expression was virtually abolished at restrictive temperatures in strains that were temperature-sensitive for RNase P (EC 3.1.26.5). Northern blot analysis showed that the steady-state copy number of EGS RNAS was several hundred per cell in vivo. A plasmid that contained,a gene for M1 RNA covalently linked to a specific EGS reduced the level of expression of a suppressor tRNA that was encoded by a separate plasmid. Similar methods can be used to regulate gene expression in E. coli and to mimic the properties of cold-sensitive mutants.

The E. coli-Streptococcus shuttle vector pDL289 can be used to introduce recombinant genes into Streptococcus sobrinus. S. sobrinus is one of the bacteria involved in dental caries. The vector was used to insert a streptococcal spectinomycin resistance gene into the scrA gene, which codes for the EIISuc permease involved in the sucrose-specific phosphoenolpyruvate phosphotransferase system in S. sobrinus.

The stable maintenance and expression of foreign genes in mollicutes (mycoplasmas) have been difficult to achieve due to the lack of suitable vectors. In this paper we show for the first time that a replicating vector can been used to express foreign genes other than antibiotic resistance genes in Acholeplasma laidlawii. Plasmids derived from the lactococcal vector pNZ18 could introduce and maintain four different genes for many generations in A. laidlawii. One of these, encoding the dominant membrane lipoprotein spiralin from the mollicute Spiroplasma citri, was expressed; however, expression was weak, the signal peptide of spiralin was not cleaved and the protein was not covalently modified by fatty acids. This resulted in a hydrophilic character of spiralin and its cytoplasmic localization in A. laidlawii. To increase the expression of foreign genes, random A. laidlawii DNA fragments were cloned into a pNZ18-related plasmid and expression signals were selected using the Bacillus licheniformis [alpha]-amylase gene as a probe. Selection was done in Escherichia coli as well as directly in A. laidlawii. Active recombinants from E. coli were also able to express [alpha]-amylase activities and an enzyme of native size in A. laidlawii. The highest activity was obtained from a recombinant selected directly in A. laidlawii. This is the first example of a promoter sequence selected in a mollicute. Analysis of the putative promoters in seven clones revealed similar - 10 and - 35 regions, and similar spacer distances in A. laidlawii, Acholeplasma oculi, Lactococcus and E. coli. Vectors related to pNZ18 should be useful for the genetic analysis of specific A. laidlawii proteins and functions.

A transposon gamma delta-containing cosmid cloning vector, pDUAL, which can be used in isolated rested deletions in large-scale and small-scale DNA sequencing, was built. pDUAL, previously termed pJANUS, uses the capacity of gamma delta to produce deletion that stretch from a tip of the transposon into nearby DNA, when the gamma delta transposes to fuse regions in the same DNA molecule.

Mycobacterial transcriptional regulation is examined by using a promoter selection vector for mycobacteria. Positive selection in mycobacteria is seen in extrachromosomal origins of Escherichia coli, and Mycobacterium fortuitum and in a kanamycin resistance gene. Mycobacterial promoter elements are detected by promoterless chloramphenicol acetyltransferase (CAT) reporter genes. Their relative strengths in a homologous environment are discovered by the genes. M. tuberculosis shows less occurrence of stronger promoters compared to M. smegmatis. The CAT actions of Escherichia coli were measured to examine the extent of divergence of mycobacterial promoters. Mycobacterial promoters which function well only in a native surrounding have been identified by the homologous selection system.

A technique for cloning yeast genes which uses a plasma vector for rescuing lambda clones as yeast centromere plasmids is presented. The system solves cloning problems caused by yeast genes' underrepresentation in genomic libraries, highly marginal phenotypes that prevent selection through genetic complementation and phenotypes requiring laborious scoring. The utility of the technique is demonstrated by using it to clone the GAL83, SSN2 and MAK7 genes.

The construction of a retroviral vector under the control of human immunodeficiency virus type 1 (HIV-1) regulatory elements and the placement of a DNA encoding endoplasmic reticulum in it were studied. Cells harvested by centrifugation from the vector were subjected to indirect immunofluorescence microscopy. The vector completely blocked the production of infectious HIV-1 virus.

The recombinant MFG retroviral vector MFG-GC was used to transduce the human gene encoding glucocerebrosidase (GC) into mouse hematopoietic stem cells (HSC). The results showed that efficiency of transfer of the GC gene to hematopoietic tissues was almost 100%.Expression of the GC gene was sustained and was several-fold higher than endogenous enzymatic activity. The results have applications for gene therapy strategies in the treatment of Gaucher disease, a lysosomal storage disease caused by deficient GC activity.

Niemann-Pick disease (NPD) is caused by deficiencies in acid sphingomyelinase (ASM). Retroviral vectors were used to transfer the normal ASM gene to type A NPD fibroblasts. The results showed that the retroviral-mediated gene transfer led to expression of ASM in these cells. This expression resulted in the reduction of accumulated sphingomyelin to normal levels. In addition, it was shown that fluorescence sorting and selective photosensitization could be used to select transduced NPD cells, without the need to preselect a marker gene.

A recombinant adenovirus with a reporter gene encoding beta-galactosidase can be used to direct efficient gene expression in myotubes derived from two specific rodent myogenic cell lines. This virus was also able to direct enzyme expression in muscle of infected mice up to 4 weeks old. Such a vector can be used in future research in bypassing muscle enzyme deficiencies in diseases such as Duchenne muscle dystrophy.

A new system for the expression of heterologous proteins in Xenopus laevis oocytes was developed. The procedure involves the injection of the viral promoter pMT2 containing the test cDNA in the nucleus of oocytes. The expression of the heterologous protein is enhanced by the coinjection of a pMT2 expression vectorcontaining a secreted alkaline phosphatase cDNA. Therefore, expressing cells can be selected by virtue of alkaline phosphatase activity. This system has been successfully tested, and thus, it is now possible to conduct protein expression studies without going through the in vitro synthesis of mRNA.

Selective substrate-host combinations were screened to select potential field application vectors. Different detergents were used as selective substrates with Pseudomonas paucimobilis 11GP4 as the host microorganism. It was found that selectivity of the substrates is proportional to their structural complexity. The more complexthe substrate structure, the more selective the substrate. This was shown by the Warburg respirometry experiments.