Stoica L, Ahmed SS, Gao G
… +1 more, Sena-Esteves M
Curr Protoc Microbiol
· 2013 · PMID 23686825
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Recombinant adeno-associated virus (rAAV) vectors are great tools for gene transfer due to their ability to mediate long-term gene expression. rAAVs have been used successfully as gene transfer vehicles in multiple anima...Recombinant adeno-associated virus (rAAV) vectors are great tools for gene transfer due to their ability to mediate long-term gene expression. rAAVs have been used successfully as gene transfer vehicles in multiple animal models of CNS disorders, and several clinical trials are currently underway. rAAV vectors have been used at various stages of development with no apparent toxicity. There are multiple ways of delivering AAV vectors to the mouse CNS, depending on the stage of development. In neonates, intravascular injections into the facial vein are often used. In adults, direct injections into target regions of the brain are achieved with great spatiotemporal control through stereotaxic surgeries. Recently, discoveries of new AAV vectors with the ability to cross the blood brain barrier have made it possible to target the adult CNS by intravascular injections.
Curr Protoc Microbiol
· 2013 Feb · PMID 23408135
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Staphylococcus aureus is an important bacterial pathogen in the hospital and community settings, especially Staphylococcus aureus clones that exhibit methicillin-resistance (MRSA). Many strains of S. aureus are utilized...Staphylococcus aureus is an important bacterial pathogen in the hospital and community settings, especially Staphylococcus aureus clones that exhibit methicillin-resistance (MRSA). Many strains of S. aureus are utilized in the laboratory, underscoring the genetic differences inherent in clinical isolates. S. aureus grows quickly at 37°C with aeration in rich media (e.g., BHI) and exhibits a preference for glycolytic carbon sources. Furthermore, S. aureus has a gold pigmentation, exhibits β-hemolysis, and is catalase and coagulase positive. The four basic laboratory protocols presented in this unit describe how to culture S. aureus on liquid and solid media, how to identify S. aureus strains as methicillin resistant, and how to generate a freezer stock of S. aureus for long-term storage.
Curr Protoc Microbiol
· 2013 Feb · PMID 23408134
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Staphylococcus aureus is a facultative anaerobic Gram-positive coccus and a member of the normal skin flora as well as the nasal passages of humans. S. aureus is also the etiological agent of suppurative abscesses, as fi...Staphylococcus aureus is a facultative anaerobic Gram-positive coccus and a member of the normal skin flora as well as the nasal passages of humans. S. aureus is also the etiological agent of suppurative abscesses, as first described by Sir Alexander Ogston in 1880. Ever since, studies on S. aureus have focused on the complex battery of virulence factors and regulators that allow for its swift transition between commensalism and pathogenic states and escape from host immune defenses. The success of this pathogen is further evidenced by its ability to acquire antibiotic resistance traits through mechanisms that often remain poorly understood.
Described in this unit are basic protocols frequently used in the research of human immunodeficiency viruses (HIVs). Provided are methods for propagating and quantifying HIV, as well as recommendations for long-term stor...Described in this unit are basic protocols frequently used in the research of human immunodeficiency viruses (HIVs). Provided are methods for propagating and quantifying HIV, as well as recommendations for long-term storage. Background information about these methods is also provided and includes their advantages, disadvantages, and troubleshooting.
Venkatesh A, Ma S, Langellotto F
… +2 more, Gao G, Punzo C
Curr Protoc Microbiol
· 2013 · PMID 23408132
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Ocular gene therapy is a fast-growing area of research. The eye is an ideal organ for gene therapy since it is immune privileged and easily accessible, and direct viral delivery results primarily in local infection. Beca...Ocular gene therapy is a fast-growing area of research. The eye is an ideal organ for gene therapy since it is immune privileged and easily accessible, and direct viral delivery results primarily in local infection. Because the eye is not a vital organ, mutations in eye-specific genes tend to be more common. To date, over 40 eye-specific genes have been identified harboring mutations that lead to blindness. Gene therapy with recombinant adeno-associated virus (rAAV) holds the promise to treat patients with such mutations. However, proof-of-concept and safety evaluation for gene therapy remains to be established for most of these diseases. This unit describes the in vivo delivery of genes to the mouse eye by rAAV-mediated gene transfer and plasmid DNA electroporation. Advantages and limitations of these methods are discussed, and detailed protocols for gene delivery, required materials, and subsequent tissue processing methods are described.
Gruntman AM, Bish LT, Mueller C
… +3 more, Sweeney HL, Flotte TR, Gao G
Curr Protoc Microbiol
· 2013 · PMID 23408131
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Adeno-associated virus (AAV) is a DNA virus with a small (∼4.7 kb) single-stranded genome. It is a naturally replication-defective parvovirus of the dependovirus group. Recombinant AAV (rAAV), for use as a gene transfer...Adeno-associated virus (AAV) is a DNA virus with a small (∼4.7 kb) single-stranded genome. It is a naturally replication-defective parvovirus of the dependovirus group. Recombinant AAV (rAAV), for use as a gene transfer vector, is created by replacing the viral rep and cap genes with the transgene of interest along with promoter and polyadenylation sequences. Only the viral inverted terminal repeats (ITRs) are required in cis for replication and packaging during production. The ITRs are also necessary and sufficient for vector genome processing and persistence during transduction. The tissue tropism of the rAAV vector is determined by the AAV capsid. In this unit we will discuss several methods to deliver rAAV in order to transduce cardiac and/or skeletal muscle, including intravenous delivery, intramuscular delivery, isolated limb infusion, intrapericardial injection in neonatal mice, and left ventricular wall injection in adult rats.
Curr Protoc Microbiol
· 2013 Feb · PMID 23408130
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Bacteria within arthropods can be identified using culture-independent methods. This unit describes protocols for surface sterilization of arthropods, DNA extraction of whole bodies and tissues, touchdown PCR amplificati...Bacteria within arthropods can be identified using culture-independent methods. This unit describes protocols for surface sterilization of arthropods, DNA extraction of whole bodies and tissues, touchdown PCR amplification using 16S rDNA general bacteria primers, and profiling the bacterial community using denaturing gradient gel electrophoresis.
Escherichia coli is a Gram-negative bacterium, commonly used in both teaching and research laboratories. This unit includes protocols for the growth and maintenance of E. coli in any teaching- or research-associated labo...Escherichia coli is a Gram-negative bacterium, commonly used in both teaching and research laboratories. This unit includes protocols for the growth and maintenance of E. coli in any teaching- or research-associated laboratory.
Selected species of the genus Agrobacterium have been extensively studied in the laboratory, but far less is known regarding their natural distribution. Agrobacterium can be isolated from a variety of environments, but i...Selected species of the genus Agrobacterium have been extensively studied in the laboratory, but far less is known regarding their natural distribution. Agrobacterium can be isolated from a variety of environments, but identifying the bacteria is challenging and involves physiological assays as well as molecular diagnostics.
This protocol was developed to utilize imaging flow cytometry (IFCM) in combination with fluorescent dyes to both enumerate and analyze morphological features of live and dead cells in a mixed live/dead bacterial sample....This protocol was developed to utilize imaging flow cytometry (IFCM) in combination with fluorescent dyes to both enumerate and analyze morphological features of live and dead cells in a mixed live/dead bacterial sample. The fluorescent dyes used in this protocol include 5(6)-carboxyfluorescein diacetate (CFDA), which indicates the functional activity of esterase inside viable bacterial cells, and DRAQ7, a dye that exploits membrane-compromised bacterial cells to enter and stain the cell. The live cell population stained with CFDA emits a fluorescent green color while the dead cell population stained with DRAQ7 emits a fluorescent red color, which allows the two populations to be distinctively separated by the IFCM system. Additionally, the cytometer captures a clear image of each object, which can then be analyzed for morphology features. The IFCM system is able to reliably, accurately, and precisely determine a bacterial cell concentration as long as the concentration of cells in a sample is no less than 1 × 10(3) cells/ml. The two dyes, CFDA and DRAQ7, have been demonstrated to be an effective stain combination for bacterial viability analysis.
Dengue is a disease caused by infection with one of the four dengue virus serotypes (DENV-1, -2, -3, and -4). The virus is transmitted to humans by Aedes sp. mosquitoes. This enveloped virus contains a positive single-st...Dengue is a disease caused by infection with one of the four dengue virus serotypes (DENV-1, -2, -3, and -4). The virus is transmitted to humans by Aedes sp. mosquitoes. This enveloped virus contains a positive single-stranded RNA genome. Clinical manifestations of dengue can have a wide range of outcomes varying from a mild febrile illness to a life-threatening condition. New techniques have largely replaced the use of DENV isolation in disease diagnosis. However, virus isolation still serves as the gold standard for detection and serotyping of DENV and is common practice in research and reference laboratories where clinical isolates of the virus are characterized and sequenced, or used for a variety of research experiments. Isolation of DENV from clinical samples can be achieved in mammalian and mosquito cells or by inoculation of mosquitoes. The experimental methods presented here describe the most common procedures used for the isolation, serotyping, propagation, and quantification of DENV.
Curr Protoc Microbiol
· 2012 Nov · PMID 23184593
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Chlamydia trachomatis is an obligate intracellular human pathogen, which lacks a system that allows genetic manipulation. Therefore, chlamydial researchers must manipulate the host cell to better understand chlamydial bi...Chlamydia trachomatis is an obligate intracellular human pathogen, which lacks a system that allows genetic manipulation. Therefore, chlamydial researchers must manipulate the host cell to better understand chlamydial biology. Host-derived lipid acquisition is critical for chlamydial survival within the host. Hence, the ability to track and purify sphingolipids in/from chlamydial infected cells has become an integral part of pivotal studies in chlamydial biology. This unit outlines protocols that provide details about labeling eukaryotic cells with exogenous lipids to examine Golgi-derived lipid trafficking to the chlamydial inclusion and then performing imaging studies or lipid extractions for quantification. Details are provided to allow these protocols to be applied to subconfluent, polarized, or siRNA knockdown cells. In addition, one will find important experimental design considerations and techniques. These methods are powerful tools to aid in the understanding of mechanisms, which allow C. trachomatis to manipulate and usurp host cell trafficking pathways.
Kuczynski J, Stombaugh J, Walters WA
… +3 more, González A, Caporaso JG, Knight R
Curr Protoc Microbiol
· 2012 Nov · PMID 23184592
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QIIME (canonically pronounced "chime") is a software application that performs microbial community analysis. It is an acronym for Quantitative Insights Into Microbial Ecology, and has been used to analyze and interpret n...QIIME (canonically pronounced "chime") is a software application that performs microbial community analysis. It is an acronym for Quantitative Insights Into Microbial Ecology, and has been used to analyze and interpret nucleic acid sequence data from fungal, viral, bacterial, and archaeal communities. The following protocols describe how to install QIIME on a single computer and use it to analyze microbial 16S sequence data from nine distinct microbial communities.
Bdellovibrio and like organisms (BALOs) are obligate predators of Gram-negative bacteria. BALOs are isolated as plaques growing at the expense of their prey and are cultivated as two-member cultures. The growth cycle is...Bdellovibrio and like organisms (BALOs) are obligate predators of Gram-negative bacteria. BALOs are isolated as plaques growing at the expense of their prey and are cultivated as two-member cultures. The growth cycle is composed of an extracellular attack phase and an intraperiplasmic elongation and replication phase. However, there are methods for obtaining host-independent (HI) mutants that grow without prey on rich media. BALOs are commonly found in the environment but generally constitute small populations; therefore, their isolation may require enrichment steps. Contamination by other bacteria during isolation necessitates efficient separation between the smaller BALO cells from the majority of larger bacteria. BALOs can also be directly detected and quantified in environmental samples using specific PCR. Synchronous cultures of both wild-type and HI derivatives can be obtained to study the different growth phases. These can be further separated by centrifugation. Classification is based on 16S rDNA analysis. Protocols relevant to these aspects of BALO detection, isolation, growth, classification, and quantitation are presented in this unit.
Huq A, Haley BJ, Taviani E
… +3 more, Chen A, Hasan NA, Colwell RR
Curr Protoc Microbiol
· 2012 Aug · PMID 22875567
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Recent molecular advances in microbiology have greatly improved the detection of bacterial pathogens in the environment. These improvements and a downward trend in the cost of molecular detection methods have contributed...Recent molecular advances in microbiology have greatly improved the detection of bacterial pathogens in the environment. These improvements and a downward trend in the cost of molecular detection methods have contributed to increased frequency of detection of pathogenic microorganisms where traditional culture-based detection methods have failed. Culture methods also have been greatly improved, and the confluence of the two suites of methods provides a powerful tool for detection, isolation, and characterization of pathogens. While molecular detection provides data on the presence and type of pathogens, culturing methods allow a researcher to preserve the organism of interest for "-omics" studies, such as genomic, metabolomic, secretomic, and transcriptomic analysis, which are rapidly becoming more affordable. This has yielded a clearer understanding of the ecology and epidemiology of microorganisms that cause disease. In this unit, we present commonly accepted methods for isolation, detection, and characterization of V. cholerae, providing more extensive knowledge of the ecology and epidemiology of this organism. This unit has been fully revised and updated from the earlier version with the latest knowledge and additional information not previously included.
Curr Protoc Microbiol
· 2012 Aug · PMID 22875566
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Adeno-associated virus (AAV) is a small replication-deficient DNA virus belonging to the Parvovirinae family. It has a single-stranded ∼4.7-kb genome. Recombinant AAV (rAAV) is created by replacing the viral rep and cap...Adeno-associated virus (AAV) is a small replication-deficient DNA virus belonging to the Parvovirinae family. It has a single-stranded ∼4.7-kb genome. Recombinant AAV (rAAV) is created by replacing the viral rep and cap genes with the transgene of interest along with promoter and polyadenylation sequences. The short viral inverted terminal repeats must remain intact for replication and packaging in production, as well as vector genome processing and persistence in the transduction process. The AAV capsid (serotype) determines the tissue tropism of the rAAV vector. In this unit we will discuss serotype selection for lung targeting along with the factors effecting efficient delivery of rAAV vectors to the murine lung. Detailed procedures for lung delivery (intranasal, orotracheal, and surgical tracheal injection), sample collection, and post-mortem tissue processing will be described.
Mueller C, Ratner D, Zhong L
… +2 more, Esteves-Sena M, Gao G
Curr Protoc Microbiol
· 2012 Aug · PMID 22875565
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In this unit, we describe the detailed procedure for a three-plasmid transfection method for rAAV production, and discuss its advantages, limitations, and troubleshooting techniques. We further discuss the rAAV purificat...In this unit, we describe the detailed procedure for a three-plasmid transfection method for rAAV production, and discuss its advantages, limitations, and troubleshooting techniques. We further discuss the rAAV purification process using CsCl gradients, as well as subsequent quality control methods using SDS-PAGE and real-time PCR to assess vector purity, packaging efficiency, and viral titer. Finally, we elaborate on a PCR-based strategy that can be used to discover novel AAV capsid sequences from primate tissue, which can be used to develop newer-generation rAAVs with a greater diversity of tissue tropism for clinical gene therapy.
The global analysis of changes in the protein composition of bacterial cells in response to treatment with antibiotic agents grants insights into the physiological response of cells to inhibition of vital cellular functi...The global analysis of changes in the protein composition of bacterial cells in response to treatment with antibiotic agents grants insights into the physiological response of cells to inhibition of vital cellular functions. This unit gives an overview of how global proteomic studies can impact antibacterial drug discovery by identifying or validating compound mechanism of action and by increasing the confidence in the value of genes with unknown function as potential new targets. It describes the design and function of a reference compendium of proteomic responses to inhibition of vital cellular functions through antibacterial agents or genetic down-regulation of potential target genes. An overview of the workflow for two-dimensional gel electrophoresis-based experiments is also presented.
Curr Protoc Microbiol
· 2012 May · PMID 22549165
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Pseudomonas aeruginosa is a common, free-living, Gram-negative bacterium that can cause significant disease as an opportunistic pathogen. Rapid growth, facile genetics, and a large suite of virulence-related phenotypes m...Pseudomonas aeruginosa is a common, free-living, Gram-negative bacterium that can cause significant disease as an opportunistic pathogen. Rapid growth, facile genetics, and a large suite of virulence-related phenotypes make P. aeruginosa a common model organism to study Gram-negative opportunistic pathogens and basic microbiology. This unit describes the basic laboratory growth and maintenance of P. aeruginosa.
Curr Protoc Microbiol
· 2012 May · PMID 22549164
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Agrobacterium species are plant-associated relatives of the rhizobia. Several species cause plant diseases such as crown gall and hairy root, although there are also avirulent species. A. tumefaciens is the most intensiv...Agrobacterium species are plant-associated relatives of the rhizobia. Several species cause plant diseases such as crown gall and hairy root, although there are also avirulent species. A. tumefaciens is the most intensively studied species and causes crown gall, a neoplastic disease that occurs on a variety of plants. Virulence is specified by large plasmids, and in the case of A. tumefaciens this is called the Ti (tumor-inducing) plasmid. During pathogenesis virulent agrobacteria copy a segment of the Ti plasmid and transfer it to the plant, where it subsequently integrates into the plant genome, and expresses genes that result in the disease symptoms. A. tumefaciens has been used extensively as a plant genetic engineering tool and is also a model microorganism that has been well studied for host-microbe associations, horizontal gene transfer, cell-cell communication, and biofilm formation. This unit describes standard protocols for simple phenotypic characterizations of A. tumefaciens.