Over the past decade, lentiviral vectors have emerged as powerful tools for transgene delivery. The use of lentiviral vectors has become commonplace and applications in the fields of neuroscience, hematology, developmental biology, stem cell biology and transgenesis are rapidly emerging. Also, lentiviral vectors are at present being explored in the context of human clinical trials. Here we describe improved protocols to generate highly concentrated lentiviral vector pseudotypes involving different envelope glycoproteins. In this protocol, vector stocks are prepared by transient transfection using standard cell culture media or serum-free media. Such stocks are then concentrated by ultracentrifugation and/or ion exchange chromatography, or by precipitation using polyethylene glycol 6000, resulting in vector titers of up to 10(10) transducing units per milliliter and above. We also provide reliable real-time PCR protocols to titrate lentiviral vectors based on proviral DNA copies present in genomic DNA extracted from transduced cells or on vector RNA. These production/concentration methods result in high-titer vector preparations that show reduced toxicity compared with lentiviral vectors produced using standard protocols involving ultracentrifugation-based methods. The vector production and titration protocol described here can be completed within 8 d.

The opportunistic human pathogen Pseudomonas aeruginosa produces a variety of virulence factors, including exotoxin A, elastase, alkaline protease, alginate, phospholipases, and extracellular rhamnolipids. The previously characterized rhlABR gene cluster encodes a regulatory protein (RhIR) and a rhamnosyltransferase (RhLAB), both of which are required for rhamnolipid synthesis. Another gene, rhlI, has now been identified downstream of the rhlABR gene cluster. The putative RhlI protein shares significant sequence similarity with bacterial autoinducer synthetases of the Luxl type. A P. aeruginosa rhilI mutant strain carrying a disrupted rhlI gene was unable to produce rhamnolipids and lacked rhamnosyltransferase activity. Rhamnolipid synthesis was restored by introducing a wild-type rhlI gene into such strains or, alternatively, by adding either the cell-free spent supernatant from a P. aeruginosa wild-type strain or synthetic N-acylhomoserine lactones. Half-maximal induction of rhamnolipid synthesis in the rhilI mutant strain required 0.5 ,IM N-butyrythomoserine lactone or 10 ,uM N-(3-oxohexanoyl)homoserine lactone. The P. aeruginosa rhUA promoter was active in the heterologous host Pseudomonas putida when both the rhiR and rhil genes were present or when the rhlR gene alone was supplied together with synthetic N-acylhomoserine lactones. The RhlR-Rhll regulatory system was found to be essential for the production of elastase as well, and crosscommunication between the RhlR-RhlI rhamnolipid regulatory system and the LasR-LasI elastase regulatory system was demonstrated.

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The host range of retroviral vectors including lentiviral vectors can be expanded or altered by a process known as pseudotyping. Pseudotyped lentiviral vectors consist of vector particles bearing glycoproteins (GPs) derived from other enveloped viruses. Such particles possess the tropism of the virus from which the GP was derived. For example, to exploit the natural neural tropism of rabies virus, vectors designed to target the central nervous system have been pseudotyped using rabies virusderived GPs. Among the first and still most widely used GPs for pseudotyping lentiviral vectors is the vesicular stomatitis virus GP (VSV-G), due to the very broad tropism and stability of the resulting pseudotypes. Pseudotypes involving VSV-G have become effectively the standard for evaluating the efficiency of other pseudotypes. This review samples a few of the more prominent examples from the ever-expanding list of published lentiviral pseudotypes, noting comparisons made with pseudotypes involving VSV-G in terms of titer, viral particle stability, toxicity, and host-cell specificity. Particular attention is paid to publications of successfully targeting a specific organ or cell types.

A mutant strain (65E12) of Pseudomonas aeruginosa that is unable to produce rhamnolipid biosurfactants and lacks rhamnosyltransferase activity was genetically complemented by using a P. aeruginosa PG201 wild-type gene library. A single complementing cosmid was isolated on the basis of surface tension measurements of subcultures of the transconjugants by using a sib selection strategy. The subcloning of the complementing cosmid clone yielded a 2-kb fragment capable of restoring rhamnolipid biosynthesis, rhamnosyltransferase activity, and utilization of hexadecane as a C source in mutant 65E12. The nucleotide sequence of the complementing 2-kb fragment was determined, and a single open reading frame (rhiR) of 723 bp specifying a putative 28-kDa protein (RhlR) was identified. Sequence homologies between the RhlR protein and some regulatory proteins such as LasR of P. aeruginosa, LuxR of Vibrio fischeri, RhiR of Rhizobium leguminosarum, and the putative activator 28-kDa UvrC of Escherichia coli suggest that the RhlR protein is a transcriptional activator. A putative target promoter which is regulated by the RhlR protein has been identified 2.5 kb upstream of the rhlR gene. Multiple plasmid-based rhlR gene copies had a stimulating effect on the growth of the P. aeruginosa wild-type strain in hexadecane-containing minimal medium, on rhamnolipid production, and on the production of pyocyanin chromophores. Disruption of the P. aeruginosa wild-type rhlR locus led to rhamnolipid-deficient mutant strains, thus confirming directly that this gene is necessary for rhamnolipid biosynthesis. Additionally, such PG201::'rhlR' mutant strains lacked elastase activity, indicating that the RhlR protein is a pleiotropic regulator.

The Human CD34؉ cells were transduced efficiently using HIV-1 pseudotype particles without prior stimulation with cytokines.

We describe a rapid and very sensitive methi$d for detecting proteins as antigens after their separationiin polyacrylamide/agarose composite gels, with or without sodium dodecyl sulfate. The polyacrylamide matrix is crosslinked with a reagent that can be cleaved with periodate or alkali to facilitate transfer of the protein bands to diazobenzyloxymethylpaper, where they are coupled covalently. Specific proteins are detected by autoradiography after sequential incubation with unfractionated, unlabeled specific antiserum and 125I-labeled protein A from Staphylococcus aureus. Antibody and protein A can be removed with urea and 2-mercaptoethanol, and the same paper can be probed again with a different antiserum. An antiserum specific for the simian virus 40 virion proteins VP3 and VP2 has been -repared; it does not crossreact with VP1, as demonstrated by t is method. An antiserum raised in rabbits against simian virus 40-transformed rabbit kidney cells is shown to be directed primarily against a periodate-sensitive moiety present in tumor (T) antigen from infected or transformed cells, whereas an antiserum raised in rabbits against large T antigen purified from lytically Specific proteins have been detected in crude extracts by immunoprecipitation with specific antisera, followed by gel electrophoresis in the presence of sodium dodecyl sulfate (NaDodSO4). The proteins are usually tagged with a radioisotope to facilitate detection and to distinguish them from the unlabeled proteins of the antisera. Studies of the tumor (T) antigens coded for by SV40 (1, 2) or polyoma (3, 4) provide recent examples of the use of this method. if the antigen is a member of a complex aggregate of different polypeptides, the entire aggregate will be precipitated and detected. Also, proteolytic degradation of the antigens in a crude extract can sometimes be a problem during the long incubation times necessary for complete immunoprecipitation. To circumvent complications such as these, alternative approaches were developed in which unlabeled proteins were first separated by gel electrophoresis and then detected by incubating the gel with radioactive antibody (5-7) or by transferring the bands of protein from the gel into an overlay of agarose impregnated with unlabeled serum (8). However, because careful washing is required to obtain good ratios of signal to background, these procedures are slow.We now describe a modification of the approach of separating the unlabeled proteins first. The procedure is simple, rapid, and very sensitive, and it gives excellent ratios of signal to background. By using this procedure, we show that SV40 T antigen has periodate-sensitive groups which can be major antigenic determinants and that an antiserum directed against the purified SV40 virion protein VP3 does not crossreact with VP1.MATERIALS AND METHODS Cells and Viruses. The CV-1 line of African monkey kidney cells and SV40 strain VA 45-54 (9) were used. SV40 virions were purified according to Christiansen et al. (10). TRK-54, an SV40-transforme...

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