Cesium Chloride Gradient Research Articles

Purification of Bacteriophages Using Anion-Exchange Chromatography.

In bacteriophage research and therapy, most applications ask for highly purified phage suspensions. The standard technique for this is ultracentrifugation using cesium chloride gradients. This technique is cumbersome, elaborate and expensive. Moreover, it is unsuitable for the purification of large quantities of phage suspensions.The protocol described here, uses anion-exchange chromatography to bind phages to a stationary phase. This is done using an FLPC system, combined with Convective Interaction Media (CIM®) monoliths. Afterward, the column is washed to remove impurities from the CIM® disk. By using a buffer solution with a high ionic strength, the phages are subsequently eluted from the column and collected. In this way phages can be efficiently purified and concentrated.This protocol can be used to determine the optimal buffers, stationary phase chemistry and elution conditions, as well as the maximal capacity and recovery of the columns.

34. Development and Validation of Identity and Homogeneity Assays for AAV Preparations

Adeno-associated virus (AAV) vectors have emerged as key clinical candidates for gene therapy. Yet, the efficiency and safety of these 20-25 nm biological nanoparticles remain difficult to harmonize across pre-clinical studies due to the limitations of current analytical tools. The presence of residual DNA, protein contaminants, empty particles and VP subunits resulting from incomplete capsid assembly are variables that can strongly modulate the reliability of in vivo data and that, therefore, need to be closely monitored in AAV research laboratories. In this work, 70 AAV preparations, obtained with various production (baculo/Sf9 and triple transfection system) and purification (iodixanol gradient and double cesium-chloride gradient) techniques were analyzed using a thermal shift assay based on the fluorescent dye Sypro® Orange. The fluorescence fingerprint obtained did not only allow to discriminate various AAV serotypes based on their capsid melting temperatures, but also enabled to probe the homogeneity and purity of AAV vector preparations, investigated in parallel using dynamic light scattering (DLS) and polyacrylamide gel electrophoresis. In particular, a double fluorescence transition indicated the presence of capsid-associated protein contaminants whereas a high initial fluorescence background correlated with the presence of free protein contaminants and capsid subunits, possibly resulting from capsid degradation during vector purification or storage. The variability, sensitivity and precision of this assay were further investigated in two different AAV research laboratories. This simple, fast (analysis of 94 preps in ~6 hrs) and low-cost assay emerges as a relevant tool for characterization of AAV vector preparations and will help to increase the reliability of in vivo gene transfer studies.

Cloning and Large-Scale Production of High-Capacity Adenoviral Vectors Based on the Human Adenovirus Type 5.

High-capacity adenoviral vectors (HCAdV) devoid of all viral coding sequences represent one of the most advanced gene delivery vectors due to their high packaging capacity (up to 35 kb), low immunogenicity and low toxicity. However, for many laboratories the use of HCAdV is hampered by the complicated procedure for vector genome construction and virus production. Here, a detailed protocol for efficient cloning and production of HCAdV based on the plasmid pAdFTC containing the HCAdV genome is described. The construction of HCAdV genomes is based on a cloning vector system utilizing homing endonucleases (I-CeuI and PI-SceI). Any gene of interest of up to 14 kb can be subcloned into the shuttle vector pHM5, which contains a multiple cloning site flanked by I-CeuI and PI-SceI. After I-CeuI and PI-SceI-mediated release of the transgene from the shuttle vector the transgene can be inserted into the HCAdV cloning vector pAdFTC. Because of the large size of the pAdFTC plasmid and the long recognition sites of the used enzymes associated with strong DNA binding, careful handling of the cloning fragments is needed. For virus production, the HCAdV genome is released by NotI digest and transfected into a HEK293 based producer cell line stably expressing Cre recombinase. To provide all adenoviral genes for adenovirus amplification, co-infection with a helper virus containing a packing signal flanked by loxP sites is required. Pre-amplification of the vector is performed in producer cells grown on surfaces and large-scale amplification of the vector is conducted in spinner flasks with producer cells grown in suspension. For virus purification, two ultracentrifugation steps based on cesium chloride gradients are performed followed by dialysis. Here tips, tricks and shortcuts developed over the past years working with this HCAdV vector system are presented.

Construction and identification of competitive 0610009E02Rik long noncoding RNA recombinant adenovirus shuttle vector

Objective To construct a competitive adenovirus vector against 0610009E02Rik, a long noncoding RNA (lncRNA) for investigating its role in the repairment of injured hepatocytes during hepatic veno-occlusive disease (HVOD). Methods Firstly, genome DNA was extracted from the tail of C57BL/6 mice, and amplified the overlapping region of the gene 0610009E02Rik and the 34th exon of Notch1 by polymerase chain reaction(RT-PCR), then it was linked into the adenovirus shuttle plasmid GV359 which was digested by BamHⅠ/NheⅠrestriction enzyme. Secondly, the constructed shuttle plasmid and packaging plasmid pBHG were co-transfected into HEK293 cells for assembly of recombination adenovirus, and subsequent amplification, followed by cesium chloride (CsCl) gradient ultracentrifugation for purification and measurement of the viral titer in HEK 293 cells. Finally, the recombination adenovirus was transfected into hepatocyte cell line H2.35 with different multiplicity of infection (MOI), and transfection efficiency was evaluated by fluorescence microscope. Meanwhile, real-time polymerase chain reaction (RT-PCR) was used to detect the expression of anti-lncRNA, Notch1 and 0610009E02Rik. Western blotting was used for the detection of the expression level of Notch1. Results ① It was discovered that the gene 0610009E02Rik and the 34th exon of Notch1 have the overlapping region (its fragment length is about 1 000 bp), and then it was obtained by PCR with the primer which has the BamHⅠ/NheⅠdigestion sites, and DNA sequencing result confirmed that the competitive adenovirus shuttle plasmid against 0610009E02Rik was successfully constructed. ② At the 12th day after transfection, about 90% cells presented cytopathic effect (CPE), then all the transfected cells were obtained a large amount of virus extract. Then HEK293 cells were transfected by the condensed and purified virus with a series of dilution, and cells transfected with dilution fold of 1∶(10~1010) were all had CPE, and this is verifi the virus titers was 5.01x109 PFU/mL. ③ Different MOI with different transfection efficiency in hepatocytes was observed and transfection efficiency of hepatocytes was about 95% after 48 h with MOI of 80. Real-time PCR was adopted to detect the relative RNA expression levels. anti-0610009E02Rik lncRNA level were (3.13±0.83) folds higher than that of the control groups (P=0.047). Notch1 mRNA relative expression level were (0.38±0.08) folds higher than that of the control group (P=0.010), and 0610009E02Rik lncRNA expression level were (1.04±0.26) folds higher than that of the control groups without significantly differences (P>0.05). Moreover, the protein level of Notch1 was lower than that of control group, and this is consistent with its mRNA level. Conclusion We successfully constructed the competitive adenovirus vector against 0610009E02Rik, which could down-regulate Notch1 mRMA and protein expression in hepatocyte cell line H2.35, providing a basis for further investigation of its role in the repairment of injury hepatocytes during HVOD. Key words: RNA, long noncoding; Receptor, Notch1; Adenovirus, human; DNA, recombinant; hepatocytes

704. Long-Term Correction of Crigler-Najjar Syndrome and Scale-Up Production of an Optimized AAV8 Vector Expressing the UGT1A1 Transgene

Crigler-Najjar syndrome (CN) is an autosomal recessive rare disorder caused by mutations in the UDP-glucuronosyltransferase 1 isotype A1 (UGT1A1) gene. In severe CN, lack or reduced activity of UGT1A1 results in high levels of serum unconjugated bilirubin (UCB), which can lead to brain damage and death. Treatment of CN consists of phototherapy for 10-12 hours per day to convert UCB into soluble photoisomers without the need of conjugation, which presents several limitations and has a major impact on life quality of the patients. Liver transplantation is the only curative option for CN. The limited therapeutic options available prompted us to develop a new therapy for CN based on the transfer of a corrected copy of the UGT1A1 gene to hepatocytes.We developed an AAV8 vector optimized for the liver expression of the hUGT1A1 transgene (AAV8-hUGT1A1). Safety and efficacy of correction of total serum bilirubin (TB) levels with AAV-hUGT1A1 were demonstrated in mouse and rat models of CN at vector doses as low as 5×10 11 vector genomes (vg)/kg, a result confirmed also by the detection of conjugated bilirubin in bile of treated rats. In juvenile CN rats, long-term correction of total bilirubin levels were observed for more than 8 months following AAV8-hUGT1A1 gene transfer. In neonate Ugt1a1–/– mice, intraperitoneal delivery of the vector at doses as low as 1×10 9 vg/mouse resulted in correction of TB at 4 weeks. However, vector delivery in P2 and P4 animals resulted in lower efficiency of correction when compared with P11-transduced mice, a finding likely due to the more advanced development of the liver at later time points.Based on these data, dose finding studies were performed in CN rats, which showed a profile of liver transduction with AAV8 vectors similar to humans, i.e. lower efficiency of transduction than mice. No differences in efficacy of correction of TB were observed in male vs. female rats.A scalable process for the production of AAV8-hUGT1A1 production was established based on a protocol of triple transfection of suspension HEK293 cells, leading to high yield vector preparations with excellent purity profile. In vitro transduction assays in human hepatocytes based on Western blot for hUGT1A1 protein and in vivo studies in CN rats demonstrated that vectors produced with the suspension process have potency characteristics undistinguishable from research grade vectors produced by triple transfection of adherent cells and purified by cesium chloride gradient centrifugation.In conclusion, our data demonstrate the safety and efficacy of gene transfer for Crigler-Najjar syndrome in two relevant animal models of the disease and provide tools and a strong rationale for the translation of these results in human subjects.

Global Contributions to the Understanding of DNA Repair and Skin Cancer

Living organisms exposed to sunlight have developed mechanisms to repair the damage that UVR induces in their DNA. These DNA repair systems are present in most life forms including bacteria, yeast, rodents, and mammals. Three rare human genetic diseases have defects in the DNA repair system called nucleotide excision repair (NER). Patients with xeroderma pigmentosum (XP) have defective NER and a more than 10,000-fold increased frequency of skin cancer, whereas patients with Cockayne syndrome (CS) or with trichothiodystrophy (TTD) have NER defects without increase in cancer. These are strikingly different clinical phenotypes (Figure 1). The dramatic differences in their manifestations reflect the complexity of DNA repair and the broad scope and central role of DNA repair on preservation of diverse biological functions. This article will review the major milestones in our understanding of DNA repair and skin cancer (Table 1). These include recognition of the clinical diseases, understanding the types of damage sunlight causes in the DNA, recognition of DNA repair in different organisms, discovery of defective DNA repair in humans, cloning of multiple human DNA repair genes, and identification of UV-type mutations in skin cancer.

Cell culture extraction and purification of rabies virus nucleoprotein.

Background:Rabies is a major zoonotic viral disease and is detected using the World Health Organization standard diagnostic techniques. Rabies detection is preferably done using the fluorescent antibody technique (FAT) that provides reliable diagnosis with almost 100% accuracy for all variant strains, if a proper conjugate is used. Rabies virus nucleoprotein (NP) is the most important protein used in production of a specific diagnostic conjugate.Objectives:The aim of this study was to extract the cell-associated rabies virus NP from infected Baby Hamster Kidney cell clone (BSR) with rabies virus (Pasteur vaccine strain/PV) and purify for a future project to produce an anti-NP conjugate.Materials and Methods:Pasteur vaccine strain (PV) as the standard rabies vaccine strain with a focus-forming dose (FFD) of 105 was inoculated in to the BSR cell culture at a concentration of 106 cells per milliliter. Infected cells were harvested 72 hours after infection and the rabies NP was extracted from these cells by low-speed centrifugation and purification by ultracentrifugation in cesium chloride (CsCl) gradient. For analysis, the purified NP was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).Results:The volume of the lysate was 15 mL and it became 2.5 mL after purification, with a concentration of 3.25 mg/mL. The corresponding band to the cell lysate protein on the SDS-PAGE had a molecular weight of 50 KDa, similar to the molecular weight of NP in rabies virus.Conclusions:The rabies virus NP could be extracted and purified in an appropriate amount from infected cell culture. The results of SDS-PAGE analysis showed that the intact rabies virus NP had been purified properly and thus could be used for further steps to produce the specific diagnostic rabies conjugate.

P38δ Regulates p53 to Control p21Cip1 Expression in Human Epidermal Keratinocytes

PKCδ suppresses keratinocyte proliferation via a mechanism that involves increased expression of p21(Cip1). However, the signaling mechanism that mediates this regulation is not well understood. Our present studies suggest that PKCδ activates p38δ leading to increased p21(Cip1) promoter activity and p21(Cip1) mRNA/protein expression. We further show that exogenously expressed p38δ increases p21(Cip1) mRNA and protein and that p38δ knockdown or expression of dominant-negative p38 attenuates this increase. Moreover, p53 is an intermediary in this regulation, as p38δ expression increases p53 mRNA, protein, and promoter activity, and p53 knockdown attenuates the activation. We demonstrate a direct interaction of p38δ with PKCδ and MEK3 and show that exogenous agents that suppress keratinocyte proliferation activate this pathway. We confirm the importance of this regulation using a stratified epidermal equivalent model, which mimics in vivo-like keratinocyte differentiation. In this model, PKCδ or p38δ knockdown results in reduced p53 and p21(Cip1) levels and enhanced cell proliferation. We propose that PKCδ activates a MEKK1/MEK3/p38δ MAPK cascade to increase p53 levels and p53 drives p21(Cip1) gene expression.

Proteomics analysis of co-purifying cellular proteins associated with rAAV vectors.

Recombinant adeno-associated vectors (rAAV) are commonly purified by either chromatography or equilibrium CsCl gradient. Nevertheless, even after purification various cellular proteins often associate with rAAV vector capsids. Such co-purifying cellular proteins may raise concern about safety of gene therapy. Here we report identification and characterization of the co-purifying cellular protein in the vector preparations by using a combination of two proteomics approaches, GeLC-MS (gel electrophoresis liquid chromatography-mass spectrometry) and 2DE (two-dimensional gel electrophoresis). Most prominent bands revealed by Coomassie Blue staining were mostly similar to the AAV capsid proteins. Posttranslational modifications of capsid proteins were detected by the proteomics analysis. A total of 13 cellular proteins were identified in the rAAV vectors purified by two rounds of cesium chloride gradient centrifugation, including 9 by the GeLC-MS analysis and 4 by the 2DE analysis. Selected cellular proteins were verified by western blot. Furthermore, the cellular proteins could be consistently found associated with different AAV serotypes and carrying different transgenes. Yet, the proteins were not integral components of the viral capsis since a stringent washing procedure by column purification could remove them. These co-purified proteins in AAV vector preparations may have a role in various stages of the AAV life cycle.

Production and Purification of Non Replicative Canine Adenovirus Type 2 Derived Vectors

Adenovirus (Ad) derived vectors have been widely used for short or long-term gene transfer, both for gene therapy and vaccine applications. Because of the frequent pre-existing immunity against the classically used human adenovirus type 5, canine adenovirus type 2 (CAV2) has been proposed as an alternative vector for human gene transfer. The well-characterized biology of CAV2, together with its ease of genetic manipulation, offer major advantages, notably for gene transfer into the central nervous system, or for inducing a wide range of protective immune responses, from humoral to cellular immunity. Nowadays, CAV2 represents one of the most appealing nonhuman adenovirus for use as a vaccine vector. This protocol describes a simple method to construct, produce and titer recombinant CAV2 vectors. After cloning the expression cassette of the gene of interest into a shuttle plasmid, the recombinant genomic plasmid is obtained by homologous recombination in the E. coli BJ5183 bacterial strain. The resulting genomic plasmid is then transfected into canine kidney cells expressing the complementing CAV2-E1 genes (DK-E1). A viral amplification enables the production of a large viral stock, which is purified by ultracentrifugation through cesium chloride gradients and desalted by dialysis. The resulting viral suspension routinely has a titer of over 10(10) infectious particles per ml and can be directly administrated in vivo.

Interleukin-12 gene adjuvant increases the immunogenicity of virus-like particles of human papillomavirus type 16 regional variant strain

ObjectivesTo analyze the immunogenicity of virus-like particles (VLP) of human papillomavirus type 16 (HPV16) isolated in East China and the adjuvant potential of interleukin-12 (IL-12). MethodsThe variant HPV16 L1VLP expressed in sf9 insect cells were purified with cesium chloride gradient centrifugation. BALB/c mice were vaccinated with VLP (L1N), VLP with Freund's adjuvant (L1A) or VLP with IL-12 recombinant plasmid (L1P). HPV16 VLP specific IgG and IFN-γ level in the serum were detected by ELISA, and the percentage of CD4+ and CD8+ in spleen cells was detected with flow cytometry. ResultsThe titers of serum IgG antibodies in vaccinated groups were higher than in negative control and the serum antibodies mainly recognized conformation-dependent HPV16 VLP epitopes. Splenic CD4+ and CD8+ T cell subsets increased after vaccination in every experimental group, and CD8+ increased obviously in L1P group. The ratio of CD4+/CD8+ decreased in L1P group and increased in the other two groups, compared to control group. Vaccination induced specific secretion of IFN-γ in the serum of vaccinated group (p<0.05), especially in the L1P group. ConclusionsVLP of HPV16 variant strain isolated in East China could induce humoral immunity and cellular immunity in mice, and IL-12 recombinant plasmid can enhance cellular immunity.

Assembly of a Marine Viral Metagenome after Physical Fractionation

Metagenomic analyses of marine viruses generate an overview of viral genes present in a sample, but the percentage of the resulting sequence fragments that can be reassembled is low and the phenotype of the virus from which a given sequence derives is usually unknown. In this study, we employed physical fractionation to characterize the morphological and genomic traits of a subset of uncultivated viruses from a natural marine assemblage. Viruses from Kāne‘ohe Bay, Hawai‘i were fractionated by equilibrium buoyant density centrifugation in a cesium chloride (CsCl) gradient, and one fraction from the CsCl gradient was then further fractionated by strong anion-exchange chromatography. One of the fractions resulting from this two-dimensional separation appeared to be dominated by only a few virus types based on genome sizes and morphology. Sequences generated from a shotgun clone library of the viruses in this fraction were assembled into significantly more numerous contigs than have been generated with previous metagenomic investigations of whole DNA viral assemblages with comparable sequencing effort. Analysis of the longer contigs (up to 6.5 kb) assembled from our metagenome allowed us to assess gene arrangement in this subset of marine viruses. Our results demonstrate the potential for physical fractionation to facilitate sequence assembly from viral metagenomes and permit linking of morphological and genomic data for uncultivated viruses.

Impact of the virus purification protocol on aggregation and electrokinetics of MS2 phages and corresponding virus-like particles

Previous experimental and theoretical studies have established that electrokinetic and aggregation properties of soft MS2 phages are not only governed by the physico-chemical features of their proteinaceous outer surface but are also significantly impacted by those of their inner RNA component (Dika et al. Appl. Environ. Microbiol., 2011, 14, 4939-4948). These conclusions contradict the recent findings of Nguyen et al. (Soft Matter, 2011, 7, 10449-10456) who reported identical electrokinetic and aggregation characteristics for MS2 and corresponding virus like particles (VLPs) that lack the internal RNA component. We demonstrate here that this contradiction originates from the different purification methods adopted prior to measurements. More generally, we show that stability and electrohydrodynamics of viruses differ according to purification by (i) dialysis, (ii) isopycnic centrifugation in the cesium chloride gradient, and (iii) precipitation using polyethylene glycol (PEG). Methods (i) and (iii) lead to aggregation of MS2 phages at pH ≤ 4 and pH ≤ 6 in 1-100 mM NaNO3 solutions, respectively, while under such conditions aggregation is not observed for MS2 and VLP suspensions prepared according to (ii). In addition, VLPs prepared following methods (i) and (iii) aggregate only at the isoelectric point (pH ~ 3-4) in 1 mM NaNO3 solution. Electrophoretic mobility data of stable MS2 and VLP particles were further examined using a recent formalism for electrokinetics of soft multilayered colloids. The analysis qualitatively shows how the purification protocol may affect either the outer particle surface properties and/or the inner particle content. Finally, the non-DLVO aggregation behavior of MS2 and VLPs purified via the above protocols is discussed in terms of the possible change in corresponding interparticular interactions.

Role of JC virus agnoprotein in virion formation

JC virus (JCV) belongs to the polyomavirus family of double-stranded DNA viruses and causes progressive multifocal leukoencephalopathy in humans. JCV encodes early proteins (large T antigen, small T antigen, and T' antigen) and four late proteins (agnoprotein, and three viral capsid proteins, VP1, VP2, and VP3). In the current study, a novel function for JCV agnoprotein in the morphogenesis of JC virion particles was identified. It was found that mature virions of agnoprotein-negative JCV are irregularly shaped. Sucrose gradient sedimentation and cesium chloride gradient ultracentrifugation analyses revealed that the particles of virus lacking agnoprotein assemble into irregularly sized virions, and that agnoprotein alters the efficiency of formation of VP1 virus-like particles. An in vitro binding assay and immunocytochemistry revealed that agnoprotein binds to glutathione S-transferase fusion proteins of VP1 and that some fractions of agnoprotein colocalize with VP1 in the nucleus. In addition, gel filtration analysis of formation of VP1-pentamers revealed that agnoprotein enhances formation of these pentamers by interacting with VP1. The present findings suggest that JCV agnoprotein plays a role, similar to that of SV40 agnoprotein, in facilitating virion assembly.

Observing better transfection efficiency in primary cultured VSMCs: Comparison and development of two different protocols

Aim: In this study, we have evaluated the procedure of transient transfection efficiency of vascular smooth muscle cells (VSMCs) with Lipofectamine (Life Technologies) and FuGENE (ROCHE, FuGENE HD Reagent) transfection reagents using the pCH110 eukaryotic assay vector containing the lacZ reporter gene. We also did try these attainments to transfect VSMCs with Ras N17 DNA and affirmed our findings. Material Methods: Plasmid pcH110, which has been purified by cesium chloride gradient centrifugation, was used in all transfections as the assay vector. And c-H-Ras was observed via Western-blot technique. Results: Briefly, the transfection with FuGENE has been given the best results, comparing with Lipofectamin. Under our culture conditions for VSMCs, FuGENE transfection efficiency could be augmented by simply increasing the amount of plasmid DNA 1.5–3 times above the recommended concentration without any visible cytotoxicity. With the FuGENE reagent, optimal transfection efficiency was obtained for primary culture of VSMCs within the recommended concentrations, but at the top of the range. The results indicate that optimization of the transfection process should include plasmid DNA concentrations above the levels suggested by the manufacturers, in order to accomplish the highest transfection efficiency. And, these finding was also supported with our Western-blot results when VSMCs have been transiently transfected with Ras N17 DNA.

Production and Titering of Recombinant Adeno-associated Viral Vectors

In recent years recombinant adeno-associated viral vectors (AAV) have become increasingly valuable for in vivo studies in animals, and are also currently being tested in human clinical trials. Wild-type AAV is a non-pathogenic member of the parvoviridae family and inherently replication-deficient. The broad transduction profile, low immune response as well as the strong and persistent transgene expression achieved with these vectors has made them a popular and versatile tool for in vitro and in vivo gene delivery. rAAVs can be easily and cheaply produced in the laboratory and, based on their favourable safety profile, are generally given a low safety classification. Here, we describe a method for the production and titering of chimeric rAAVs containing the capsid proteins of both AAV1 and AAV2. The use of these so-called chimeric vectors combines the benefits of both parental serotypes such as high titres stocks (AAV1) and purification by affinity chromatography (AAV2). These AAV serotypes are the best studied of all AAV serotypes, and individually have a broad infectivity pattern. The chimeric vectors described here should have the infectious properties of AAV1 and AAV2 and can thus be expected to infect a large range of tissues, including neurons, skeletal muscle, pancreas, kidney among others. The method described here uses heparin column purification, a method believed to give a higher viral titer and cleaner viral preparation than other purification methods, such as centrifugation through a caesium chloride gradient. Additionally, we describe how these vectors can be quickly and easily titered to give accurate reading of the number of infectious particles produced.

Production and Titering of Recombinant Adeno-associated Viral Vectors

In recent years recombinant adeno-associated viral vectors (AAV) have become increasingly valuable for in vivo studies in animals, and are also currently being tested in human clinical trials. Wild-type AAV is a non-pathogenic member of the parvoviridae family and inherently replication-deficient. The broad transduction profile, low immune response as well as the strong and persistent transgene expression achieved with these vectors has made them a popular and versatile tool for in vitro and in vivo gene delivery. rAAVs can be easily and cheaply produced in the laboratory and, based on their favourable safety profile, are generally given a low safety classification. Here, we describe a method for the production and titering of chimeric rAAVs containing the capsid proteins of both AAV1 and AAV2. The use of these so-called chimeric vectors combines the benefits of both parental serotypes such as high titres stocks (AAV1) and purification by affinity chromatography (AAV2). These AAV serotypes are the best studied of all AAV serotypes, and individually have a broad infectivity pattern. The chimeric vectors described here should have the infectious properties of AAV1 and AAV2 and can thus be expected to infect a large range of tissues, including neurons, skeletal muscle, pancreas, kidney among others. The method described here uses heparin column purification, a method believed to give a higher viral titer and cleaner viral preparation than other purification methods, such as centrifugation through a caesium chloride gradient. Additionally, we describe how these vectors can be quickly and easily titered to give accurate reading of the number of infectious particles produced.

Identification of a Toluene-Degrading Bacterium from a Soil Sample through H 2 18 O DNA Stable Isotope Probing

DNA stable isotope probing (DNA-SIP) with H(2)(18)O was used to identify a toluene-degrading bacterium in soil amended with 48 ppm toluene. After quantification of toluene degradation rates in soil, DNA was extracted from soil incubated with H(2)(18)O, H(2)(16)O, H(2)(16)O and 48 ppm toluene, or H(2)(18)O and 48 ppm toluene. A single DNA band formed along a cesium chloride gradient after isopycnic centrifugation of extracts from soils incubated with H(2)(16)O. With extracts from soils to which only H(2)(18)O was added, two distinct DNA bands formed, while three bands formed when DNA extracted from soil incubated with both H(2)(18)O and toluene was analyzed. We suggest that this third band formed because toluene does not contain any oxygen atoms and toluene-degrading organisms had to transfer oxygen atoms from H(2)(18)O into metabolic intermediates to form nucleic acids de novo. We extracted the third DNA band and amplified a large fraction of the bacterial 16S rRNA gene. Direct sequencing of the PCR product obtained from the labeled DNA, as well as cloned 16S rRNA amplicons, identified a known toluene degrader, Rhodococcus jostii RHA1. A toluene-degrading bacterial strain was subsequently isolated from soil and shown to be Rhodococcus jostii RHA1. Finally, quantitative real-time PCR analysis showed that the abundance of the 16S rRNA gene of Rhodococcus jostii RHA1 increased in soil after toluene exposure but not in soils from which toluene was withheld. This study indicates that H(2)(18)O DNA-SIP can be a useful method for identifying pollutant-degrading bacteria in soil.

Cesium chloride gradient centrifugation improves the quality of total RNA preparations from the gastropod Strombus gigas and the coral Montastraea faveolata

RNA preparation from marine invertebrate samples including gastropods and cnidarians is often difficult, producing low-purity RNA samples that perform poorly in downstream procedures. This is increasingly problematic given the current interest in transcriptional studies of marine invertebrates. We report an RNA preparation method using a phenol/chloroform extraction followed by CsCl gradient centrifugation, modified from previous reports and adapted for a modern benchtop ultracentrifuge. This method produced improved spectrophotometric indicators of RNA quality (A 260/A 280 and A 260/A 230) for Strombus gigas Linnaeus, 1758, testis samples and improved RNA integrity number (RIN) for Montastraea faveolata Ellis, 1786, samples, relative to a preparation procedure that relies solely on chemical extraction of RNA. Rate of success in microarray labeling reactions was slightly, but not significantly, improved following CsCl gradient centrifugation. A real-time RT-PCR assay performed using CsCl-prepared samples was efficient (92.5%) and produced consistent and reproducible results.

Physical fractionation of aquatic viral assemblages

Aquatic viruses are extraordinarily diverse and have major influences on plankton ecology and evolution, but the majority of these viruses remain uncharacterized. Most viruses have not been cultivated and cultivationindependent means to explore viral diversity have provided only a fragmented view of viral genomic information. In this study, ultracentrifugation and chromatographic methods were evaluated for their ability to fractionate aquatic viruses based on their differing physical properties with the aim of physically enriching subsets of aquatic viruses for further study. Centrifugation in continuous cesium chloride gradients, strong and weak anion‐exchange chromatography, and size‐exclusion chromatography with Sephacryl S‐1000 were found to separate aquatic viruses based on their differing buoyant densities, surface charges, and sizes, respectively. Sucrose density gradients, cation‐exchange chromatography, and size‐exclusion chromatography using controlled‐pore glass were found to have insufficient resolution to effectively separate viral assemblages. Using the successful methods listed above, we were able to separate complex assemblages of viruses into fractions that had distinguishable subsets of the viruses present in the original community as determined by pulsed‐field gel electrophoresis. Strong anion‐exchange chromatography was particularly effective at separating viruses in the samples and resulted in one to six dominant viral genome bands in most fractions. Our results suggest that individual populations of viruses may be physically enriched, and possibly isolated, from complex assemblages without cultivation. These fractionation methods are expected to improve our ability to characterize the genotypes and phenotypes of the uncultivated majority of viruses in aquatic environments.