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Transduction of stem cells with a marking gene holds promise to determine if tissue repair or regeneration is derived from the adult hematopoietic stem cell and if relapse of an autoimmune disease should occur whether relapse arises from the stem cell compartment or from lymphocytes surviving the conditioning regimen. New safety concerns about gene-modified stem cell would entail new safety testing such as documentation of the insertional site prior to release.

Gene transfer from the chloroplast to the nucleus has occurred over evolutionary time. Functional gene establishment in the nucleus is rare, but DNA transfer without functionality is presumably more frequent. Here, we measured directly the transfer rate of chloroplast DNA (cpDNA) into the nucleus of tobacco plants (Nicotiana tabacum). To visualize this process, a nucleus-specific neomycin phosphotransferase gene (neoSTLS2) was integrated into the chloroplast genome, and the transfer of cpDNA to the nucleus was detected by screening for kanamycin-resistant seedlings in progeny. A screen for kanamycin-resistant seedlings was conducted with about 250,000 progeny produced by fertilization of wild-type females with pollen from plants containing cp-neoSTLS2. Sixteen plants of independent origin were identified and their progenies showed stable inheritance of neoSTLS2, characteristic of nuclear genes. Thus, we provide a quantitative estimate of one transposition event in about 16,000 pollen grains for the frequency of transfer of cpDNA to the nucleus. In addition to its evident role in organellar evolution, transposition of cpDNA to the nucleus in tobacco occurs at a rate that must have significant consequences for existing nuclear genes.

Direct protein delivery is an emerging technology in vaccine development and gene therapy. We could previously show that subviral dense bodies (DB) of human cytomegalovirus (HCMV), a beta-herpesvirus, transport viral proteins into target cells by membrane fusion. Thus these non-infectious particles provide a candidate delivery system for the prophylactic and therapeutic application of proteins. Here we provide proof of principle that DB can be modified genetically. A 55 kDa fusion protein consisting of the green fluorescent protein and the neomycin phosphotransferase could be packed in and delivered into cells by recombinant DB in a functional fashion. Furthermore, transfer of protein into fibroblasts and dendritic cells by DB was efficient, leading to exogenous loading of the MHC-class I antigen presentation pathway. Thus, DB may be a promising basis for the development of novel vaccine strategies and therapeutics based on recombinant polypeptides.

The misuse of antibiotics has selected for bacteria that have evolved mechanisms for evading the effects of these drugs. For aminoglycosides, a group of clinically important bactericidal antibiotics that target the A-site of the 16S ribosomal RNA, the most common mode of resistance is enzyme-catalyzed chemical modification of the drug. While aminoglycosides are structurally diverse, a single enzyme can confer resistance to many of these antibiotics. For example, the aminoglycoside kinase APH(3')-IIIa, produced by pathogenic Gram-positive bacteria such as enterococci and staphylococci, is capable of detoxifying at least 10 distinct aminoglycosides. Here we describe the crystal structures of APH(3')-IIIa in complex with ADP and kanamycin A or neomycin B. These structures reveal that the basis for this enzyme's substrate promiscuity is the presence of two alternative subsites in the antibiotic binding pocket. Furthermore, comparison between the A-site of the bacterial ribosome and APH(3')-IIIa shows that mimicry is the second major factor in dictating the substrate spectrum of APH(3')-IIIa. These results suggest a potential strategy for drug design aimed at circumventing antibiotic resistance.

Advances in the development of highly infectious, replication-deficient recombinant retroviruses provide an efficient means of stable transfer of gene expression. Coupled with ex vivo transduction, surrogate cell populations can be readily implanted into the brain, thus serving as vehicles for delivering selected gene products into the central nervous system (CNS). Here we report that rat astrocytes can be routinely and safely isolated from brain tissue of a living donor by use of short-term gelatin sponge implants. The mature, nontransformed astrocytes were easily expanded, maintained in long-term tissue cultures and were efficiently transduced with an amphotropic retrovirus harboring a heterologous, fused transgene. In vitro retroviral infection rendered the nontransformed cells essentially 100% viable after exposure. The level of efficiency of infection (30-50% effective genome integration of provirus and expression of transgene in target cell populations) and minimal cell toxicity obviated the need to harvest large numbers of target cells. Cultured transduced astrocytes were resilient and exhibited select peptide expression for up to 1 year. Subsequently, transduced astrocytes were used in a series of experiments in which cells were transplanted intracerebrally in syngeneic animals. Post-implantation, astrocytes seeded locally and either insinuated into the surrounding parenchyma in situ or exhibited a variable degree of migration, depending on the anatomic source of astrocytes and the targeted brain implantation site. Transduced astrocytes remained viable in excess of 8 months post-transplantation and exhibited sustained transgenic peptide expression of green fluorescent protein/neomycin phosphotransferase in vivo. The sequential isolation and culture of nontransformed, mature, adult astrocytes and recombinant retrovirus-mediated transduction in vitro followed by brain reimplantation represents a safe and effective means for transferring genetic expression to the CNS. This study lays the foundation for exploring the utility of using a human autologous transplantation system as a potential gene delivery approach to treat neurological disorders. Prepared and utilized in this manner, autologous astrocytes may serve as a vehicle to deliver gene therapy to the CNS.

This investigation reports a protocol for transfer and expression of foreign chimeric genes in loblolly pine (Pinus taeda L.). Transformation was achieved by co-cultivation of mature zygotic embryos with Agrobacterium tumefaciens strain LBA4404 which harbored a binary vector (pBI121) including genes for beta-glucuronidase (GUS) and neomycin phosphotransferase (NPTII). Factors influencing transgene expression including seed sources of loblolly pine, concentration of bacteria, and the wounding procedures of target explants were investigated. The expression of foreign gene was confirmed by the ability of mature zygotic embryos to produce calli in the presence of kanamycin, by histochemical assays of GUS activity, by PCR analysis, and by Southern blot. The successful expression of the GUS gene in different families of loblolly pine suggests that this transformation system is probably useful for the production of the genetically modified conifers.

The murine MBT-2 bladder tumor model in syngeneic C3H/HeN mice was used to investigate the feasibility of gene therapy based on the delivery of interferon-gamma (IFN-gamma) in vivo by retroviral vectors. We constructed a recombinant retroviral vector pRUFneo/IFN-gamma, which was transfected into a retroviral packaging cell line psiCRE, to produce psiCRE/pRUFneo/IFN-gamma cells. The expressions of the neo and IFN-gamma genes were verified by reverse transcription-polymerase chain reaction and IFN-gamma was detected in the culture supernatant from psiCRE/pRUFneo/IFN-gamma cells. After receiving MBT-2 cells admixed with retroviral pRUFneoIFN-gamma supernatant, C3H/HeN mice exhibited lower tumor incidence, lower tumor mass, and higher survival rate, as well as higher antitumor responses compared to those injected with MBT-2 cells admixed with control retroviral supernatant. Moreover, the retroviral pRUFneoIFN-gamma supernatant was able to suppress the growth of rechallenged tumors in postoperated mice. Although the IFN-gamma protein secreted from psiCRE/pRUFneo/IFN-gamma cells partly contributes to the antitumor effect of retroviral pRUFneoIFN-gamma supernatant, the retroviruses carrying the IFN-gamma gene transduced MBT-2 cells in vivo, which may result in enhancing local IFN-gamma production from tumor cells. Because bladder is suitable for the intravesical instillation of therapeutic agents, in vivo administration of retroviral vectors encoding IFN-gamma may be explored for the treatment of bladder cancer.

The purpose of this study was to determine the feasibility of cytokine gene delivery to lymphatic tissue using transduced bone marrow-derived cells. MBAE and pBABE retroviral vectors carrying the genes for murine interleukin-4 and the selection marker neomycin phosphotransferase (neo) were used to transduce bone marrow-derived dendritic cells (DC) and hematopoietic stem cells (HSC). A transduction efficiency of 11-33% for HSC and 2-10% for DC was achieved. Transduced HSC and DC released 55-170 pg of recombinant interleukin-4 per 1 x 10(6) cells/mL in vitro. To study the migration of the cells in vivo, we introduced the transduced cells into syngenic mice. DC were injected subcutaneously into the front limbs of unconditioned mice and HSC were intravenously administered to irradiated mice. The distribution of the transduced cells was studied by quantitative polymerase chain reaction for the neo gene as a marker. After 3 days, DC migrated to the axillary lymph nodes in the drainage area of the injection site and were detectable up to 5 days. After intravenous administration of transduced HSC, the neo gene could be found in up to 100 copies/5 x 10(3) cells in mesenterial lymph node, spleen, bone marrow, thymus, and liver. The distribution of the transduced cells was heterogenous: in different mice, different organs showed high copies of the neo gene after 10 and 13 days. After 39 days, two of three mice were negative for neo in all organs analyzed. In conclusion, bone marrow-derived cells can be genetically engineered ex vivo to deliver recombinant cytokine genes to lymphoid organs in vivo. In particular, DC might be candidate cells for use in immunomodulatory gene therapy for autoimmune diseases and cancer.

The faithful repair of DNA damage such as chromosomal double-strand breaks (DSBs) is crucial for genomic integrity. Aberrant repair of these lesions can result in chromosomal rearrangements, including translocations, which are associated with numerous tumours. Models predict that some translocations arise from DSB-induced recombination in differentiating lymphoid cell types or from aberrant repair of DNA damage induced by irradiation or other agents; however, a genetic system to study the aetiology of these events has been lacking. Here we use a mouse embryonic stem cell system to examine the role of DNA damage on the formation of translocations. We find that two DSBs, each on different chromosomes, are sufficient to promote frequent reciprocal translocations. The results are in striking contrast with interchromosomal repair of a single DSB in an analogous system in which translocations are not recovered. Thus, while interchromosomal DNA repair does not result in genome instability per se, the presence of two DSBs in a single cell can alter the spectrum of repair products that are recovered.

Metabolic suicide gene transfer is widely applied for gene therapy of cancer, and retroviral vectors expressing the herpes simplex virus thymidine kinase (HSV-tk) gene are commonly used in clinical trials. Most of these vectors contain positive selectable markers that undoubtedly facilitate the determination of viral titer and the identification of high-titer producer clones. However, the presence of additional transcriptional units may result in reduced expression of the gene of interest. The use of fusion genes expressing bifunctional proteins may help to overcome this problem. We have constructed a retroviral vector carrying the TNFUS69 chimeric gene, which originates from the fusion of the HSV-tk and neomycin phosphotransferase II genes, and evaluated the functional expression of the encoded fusion protein. In vitro, expression of the fusion gene conferred to target cells both resistance to neomycin and selective sensitivity to the antiherpetic drugs ganciclovir and (E)-5-(2-bromovinyl)-2'-deoxyuridine. Cells transduced with the fusion gene, however, showed reduced ability to phosphorylate ganciclovir compared with cells expressing the native HSV-tk. Therefore, although the fusion gene may be used as a constituent of retroviral cassettes for positive and negative selection in vitro, its usefulness for suicide gene transfer applications in vivo may depend upon the possibility of using (E)-5-(2-bromovinyl)-2'-deoxyuridine in a clinical context.

The repair of DNA double-strand breaks is essential for cells to maintain their genomic integrity. Two major mechanisms are responsible for repairing these breaks in mammalian cells, non-homologous end-joining (NHEJ) and homologous recombination (HR): the importance of the former in mammalian cells is well established, whereas the role of the latter is just emerging. Homologous recombination is presumably promoted by an evolutionarily conserved group of genes termed the Rad52 epistasis group. An essential component of the HR pathway is the strand-exchange protein, known as RecA in bacteria or Rad51 in yeast. Several mammalian genes have been implicated in repair by homologous recombination on the basis of their sequence homology to yeast Rad51: one of these is human XRCC2. Here we show that XRCC2 is essential for the efficient repair of DNA double-strand breaks by homologous recombination between sister chromatids. We find that hamster cells deficient in XRCC2 show more than a 100-fold decrease in HR induced by double-strand breaks compared with the parental cell line. This defect is corrected to almost wild-type levels by transient transfection with a plasmid expressing XRCC2. The repair defect in XRCC2 mutant cells appears to be restricted to recombinational repair because NHEJ is normal. We conclude that XRCC2 is involved in the repair of DNA double-strand breaks by homologous recombination.

The expression of human hepatitis B virus (HBV) surface (HBS) and e (HBe) antigens has been studied comparatively in monkey and mouse cell lines co-transfected with HBV DNA and the dominant selective marker aminoglycoside 3'-phosphotransferase gene. We have found that the kinetics and stability of expression of the HBS gene varies with the cell lines used. Only a late transient expression of both HBS and HBe is observed between 1 and 5 weeks after transfection in monkey kidney Vero cells transfected with the complete HBV genome, while a permanent expression of HBS and HBe is obtained in mouse cells. HBS and HBe are excreted into the cell culture medium. HBe is expressed in cells transfected with the complete HBV genome, but not with isolated HBS gene. In clones of Vero cells transformed with the HBS gene, HBV sequences were rearranged or lost.

The light-inducible nuclear gene coding for the small subunit of ribulose-1,5-bisphosphate carboxylase (Rubisco), produces a precursor protein with an amino-terminal transit peptide which is transported into the plastids and cleaved by a specific proteinase. To test whether the promoter and transit peptide-coding sequences of the small subunit gene can be used to direct the light-inducible synthesis and transport of a foreign protein into chloroplasts, a chimeric gene was constructed consisting of the promoter, first exon and intron as well as part of the second exon of the small subunit Rubisco gene fused to the amino-terminal end of the neomycin phosphotransferase II gene (nptII) of Tn5. Tobacco tissue, as well as whole plants, into which this chimaeric gene was introduced, were resistant to kanamycin. The transcription of the chimaeric gene as well as the NPTII activity of the resulting fusion protein were shown to be light inducible. The fusion protein is processed and located within the chloroplasts of the transformed plants.