scientificprotocols

Authors: Thomas Wurdinger , Christian Badr & Bakhos Tannous 

Introduction

Luciferase-mediated bioluminescence is widely used as a reporting tool for monitoring various biological processes in vitro and in vivo. Firefly (1), Renilla (2), and Gaussia (3,4) luciferases are currently used as reporters for monitoring of numerous processes in different fields (5), including immunology (6) oncology (7), virology (8), and neuroscience (9). After systemic substrate injection, a cooled charge-coupled device (CCD) camera can be used for the localization of the luciferase photon signals in vivo. In addition, the naturally secreted and non-ATP dependent Gaussia luciferase (Gluc) expression levels can be easily quantified in cell-free conditioned medium by adding its substrate coelenterazine and measuring emitted photons using a luminometer.

Since Gaussia luciferase is naturally secreted from mammalian cells in culture (4), it is secreted into the blood of animals harboring cells expressing this reporter

scientificprotocols

Authors: Erin Cram & Jean Schwarzbauer 

Introduction

RNAi is an effective method for analyzing gene function in C. elegans that often phenocopies loss-of-function phenotypes (1). In RNAi, double stranded RNA (dsRNA) introduced into larvae or adults activates an enzymatic pathway that eliminates endogenous RNAs homologous to the dsRNA (2). Potent and persistent RNAi silencing in results from secondary amplification of small amounts of the initial RNAi trigger by RNA dependent RNA polymerases (3, 4). RNAi can be induced in C. elegans using one of four methods: injecting in vitro synthesized dsRNA into the body cavity of the animal (injection RNAi), soaking in a solution of dsRNA (soaking RNAi), feeding animals bacteria engineered to express dsRNA (feeding RNAi), or through creation of transgenic animals that express dsRNA from DNA arrays maintained within cells (hairpin RNAi) (5, 6). This protocol is slightly modified from (7), and can be used for candidate or broad RNAi screens in C. elegans

scientificprotocols

Authors: Xuejiao Wang , Michelle Starz-Gaiano , Tina Bridges & Denise Montell 

Introduction

We developed a strategy to purify a small cell population from the Drosophila ovary for the purpose of gene expression profiling (used in Wang et al, 2006b). The Drosophila ovary is comprised of ovarioles, which are strings of egg chambers at successively later stages of development. Within each egg chamber, there are 16 large germ line cells encompassed by about 650 somatic epithelial follicle cells. Of this set of epithelial cells, some cells will take on specialized fates, such as the border cells. The border cells are a cluster of 6-8 cells that detach from the epithelium and actively migrate, as part of their normal development. Once they reach their target, a second population of follicle cells, centripetal cells, initiate migration.

We were interested in the gene expression profiles that distinguish the migratory border and centripetal cells from the non-motile somatic cells and the germ line cells t

scientificprotocols

Authors: Dennis Hallahan & Z. Han 

Introduction

Phage display is a method to discover peptide ligands while minimizing and optimizing the structure and function of proteins (Hallahan, 2003). The phage is used as a scaffold to display recombinant libraries of peptides and provides a means to recover and amplify the peptides that bind to putative receptor molecules in vivo. In vivo selection simultaneously provides positive and subtractive screens because organs and tissues such as tumors are spatially separated. Phage DNA can then be sequenced to determine the amino acid sequence of peptides on the capsid that have been recovered from tumors. The T7 phage display system exploits the T7 capsid protein as a scaffold to display peptides on the capsid protein unique to the 10B protein on the surface of the phage. Gene 10 encoding the capsid protein is cloned with a series of multiple cloning sites at the C-terminus of the 10B protein. The natural translational frame shift site within the capsid gene has

scientificprotocols

Authors: Edward Owusu-Ansah, Amir Yavari & Utpal Banerjee 

Abstract

2’,7’-dichlorofluorescein (H2DCF) and Dihydroethidium (DHE), have been used extensively in tissue culture experiments to evaluate reactive oxygen species (ROS) production. However, it will be more advantageous to be able to detect real time ROS production in live tissues, especially in Drosophila where the extensive genetic tools available make it possible to compare the phenotype of mutant tissue juxtaposed to its wild-type neighbor. Here, we describe a protocol for imaging ROS production in Drosophila using either H2DCF or DHE. We highlight the specific advantage posed by monitoring ROS production in vivo by comparing the phenotype of cells mutant for genes encoding mitochondrial proteins with their wild-type neighbors. We also show from staining of the germarium that this technique is capable of detecting different levels of ROS production among cells within the same tissue. The whole protocol, from dissection to capturing o

scientificprotocols

Authors: H. James Tripp 

Introduction

The Guava Easy-Cyte flow cytometer accepts 96 well plates containing stained cells in liquid media and reports the cells/ml for each well in Excel format (csv file). Marine microbes stained with SYBR-Grn I in natural seawater can be counted within approximately 5% error at densities of 1.0E4 to 5.5E5 cells per ml. With exceptional care, cell cultures can be reliably detected but not accurately counted down to 2E3 cells per ml. At densities above 5.5E5, cells must be diluted for accurate counting. There are a few critical adaptations to the manufacturer’s operating instructions that must be made relating to use of seawater media for extremely small SAR11 cells. Neutral pH detergent must be used for cleaning or salts will precipitate and foul the fluid pump of the cytometer. Stain concentration must be higher than recommended and made fresh daily, voltage must be higher than recommended, and rinse tubes must be changed daily. Also, freeze/thaw cycles of SYBR-Grn

scientificprotocols

Authors: Xue-Ming Xu, Min-Hyuk Yoo, Bradley Carlson & Dolph Hatfield 

Introduction

The use of RNAi technology to target the removal of mRNA expression has become a powerful tool in studying protein function (e.g., see references 1 & 2). RNAi technology has several constraints, however, and the more improvements that can be made in the procedure, the more powerful and useful the technique becomes. One drawback has been that RNAi technology has been used primarily to target the knockdown of a single gene. This technology was recently expanded to simultaneously target the knockdown of two or more genes in a variety of organisms by developing vectors for inserting multiple siRNA encoding sequences (3-6). Similarly, as described herein, we have developed a vector for simultaneously targeting the removal of multiple genes in mammalian systems. Another limitation in RNAi technology has been that it examined the role of proteins only by their loss preventing an in-depth analyses of overall function of the

scientificprotocols

Authors: Mark Howarth & Alice Ting 

Introduction

Monovalent streptavidin is a heterotetramer consisting of 3 dead subunits which do not bind biotin and one alive subunit which binds biotin with high affinity. Dead (D) and Alive (A) subunits are expressed separately in E. coli, where they form inclusion bodies. D and A inclusion bodies are dissolved in guanidinium hydrochloride, mixed in an appropriate ratio, and refolded by rapid dilution into PBS. The refold creates a statistical mixture of tetramers, and monovalent streptavidin is purified from the other tetramers by a nickel affinity column, since its single His6 tag gives it different elution properties than the tetramers with 0, 2, 3 or 4 His6 tags.

Procedure

A) Express Alive and Dead streptavidin subunits • TIMING 2 days for steps 1-2; 8 hr for steps 3-5, 3 hr for steps 6-7

scientificprotocols

Authors: Jan Utermark & Petr Karlovsky 

Abstract

Agrobacterium tumefaciens-mediated transformation of filamentous fungi consists of (i) induction of A. tumefaciens culture harbouring a binary vector, (ii) co-incubation of bacteria with fungal spores on a solid support, and (iii) selection of transformants. During the induction, vir genes on the helper component of the binary vector are activated, conditioning A. tumefaciens for transformation. During co-cultivation, T-DNA part of the binary vector system is transferred into fungal nucleus and inserted into the genome. Transformants are selected on a medium with appropriate antibiotic. In order to maximize the number of transformants, the ratio of A. tumefaciens cells to fungal spores and the duration of the co-cultivation need be optimized. The procedure takes two to three weeks for fast-growing fungi.

Introduction

Kingdom Fungi consists of about two million species (1), which include pathogens and symbionts of plants and animals and

scientificprotocols

Authors: Nikos Kourtis & Nektarios Tavernarakis 

Introduction

Currently available methodologies for measuring protein synthesis rates rely on metabolic labelling by incorporation of radioactive amino acids into nascent polypeptides. These approaches are hampered by several limitations and cannot be applied to monitor protein synthesis in specific cells or tissues, in live specimens. Here, we describe a novel method for monitoring protein synthesis in specific cells and tissues of live Caenorhabditis elegans animals. Fluorescent reporter proteins such as GFP are expressed in specific cells and tissues of interest or throughout animals using appropriate promoters. Protein synthesis rates are assessed by following fluorescence recovery after partial photobleaching of the fluorophore at targeted sites. We evaluate the method by examining protein synthesis rates in diverse cell types of the nematode. Because it is non-invasive, our approach allows monitoring of protein synthesis in single cells or tissu