Showing papers in "Current protocols in molecular biology in 2003"

Recombineering: Genetic Engineering in Bacteria Using Homologous Recombination

Abstract: The bacterial chromosome and bacterial plasmids can be engineered in vivo by homologous recombination using PCR products and synthetic oligonucleotides as substrates. This is possible because bacteriophage-encoded recombination proteins efficiently recombine sequences with homologies as short as 35 to 50 bases. Recombineering allows DNA sequences to be inserted or deleted without regard to location of restriction sites. This unit first describes preparation of electrocompetent cells expressing the recombineering functions and their transformation with dsDNA or ssDNA. It then presents support protocols that describe several two-step selection/counter-selection methods of making genetic alterations without leaving any unwanted changes in the targeted DNA, and a method for retrieving onto a plasmid a genetic marker (cloning by retrieval) from the Escherichia coli chromosome or a co-electroporated DNA fragment. Additional protocols describe methods to screen for unselected mutations, removal of the defective prophage from recombineering strains, and other useful techniques.

Helicase dependent amplification of nucleic acids

Abstract: Methods and a kit are provided for selectively and exponentially amplifying nucleic acids and include the use of a helicase preparation and a DNA polymerase such that the amplification can be performed isothermally.

Calcium phosphate transfection

Abstract: This unit presents two methods of calcium phosphate-based eukaryotic cell transfection that can be used for both transient and stable transfections. In these protocols, plasmid DNA is introduced to monolayer cell cultures via a precipitate that adheres to the cell surface. A HEPES-buffered solution is used to form a calcium phosphate precipitate that is directly layered onto the cells. For some cells, shocking the cells with glycerol or DMSO improves transfection efficiency. In the alternate high-efficiency method, a BES-buffered system is used that allows the precipitate to form gradually in the medium and then drop onto the cells. While the alternate method is particularly efficient for stable transformation of cells with circular plasmid DNA, both protocols yield similar results for transformation with linear plasmid or genomic DNA, or for transient expression.

RNA interference in Caenorhabditis elegans.

Abstract: RNAi has become an essential tool in C. elegans research. Procedures for RNAi in C. elegans by microinjecting with dsRNA, feeding with bacteria expressing dsRNA and soaking in dsRNA solution are described in this unit.

Transfection by electroporation.

Abstract: Electroporation-the use of high-voltage electric shocks to introduce DNA into cells-can be used with most cell types, yields a high frequency of both stable transformation and transient gene expression, and, because it requires fewer steps, can be easier than alternate techniques. This unit describes electroporation of mammalian cells, including ES cells for the preparation of knock-out, knock-in, and transgenic mice. Protocols are described for the use of electroporation in vivo to perform gene therapy for cancer therapy and DNA vaccination. Also described are modifications for preparation and transfection of plant protoplasts. © 2018 by John Wiley & Sons, Inc.

Cloning of Small RNA Molecules

Abstract: Small RNAs that are derived from dsRNA precursors act as guide RNAs during sequence-specific epigenetic regulation of eukaryotic gene expression. These small regulatory RNAs are between 20 and 30 nucleotides in length, and fall into one or more of the following categories: small interfering RNAs (siRNAs), microRNAs (miRNAs), and heterochromatic siRNAs (hsiRNAs). Procedures to record the profile of small RNAs expressed in cultured cells or tissues are described. The small RNAs are directionally cloned after isolation from total RNA. The methods rely on T4 RNA ligase-based joining of adapter oligonucleotides to the 3' and 5' termini of the pool of small RNAs. The ligation products are reverse transcribed and PCR-amplified. It is recommended to directionally concatamerize the relatively short PCR products before cloning in order to increase the number of RNA sequences obtained per clone.

Staining Proteins in Gels

Abstract: This unit describes protocols for detecting protein in a gel by either Coomassie blue staining or silver staining. The former is easier and more rapid; however, silver staining methods are considerably more sensitive and thus can be used to detect smaller amounts of protein. Alternate rapid staining procedures are provided for each method and a support protocol describes how to photograph stained gels. Fluorescent staining is a popular alternative to traditional staining procedures, mainly because it is more sensitive than Coomassie staining, and often as sensitive as silver staining. Staining of proteins in SDS-polyacrylamide gels is described, and an alternate protocol details variations in the procedure for proteins in nondenaturing gels. A final support protocol describes the photography of fluorescently stained proteins.

Selection of transfected mammalian cells

Abstract: Analysis of gene function frequently requires the formation of mammalian cell lines that contain the studied gene in a stably integrated form. Approximately one in 10(4) cells in a transfection will stably integrate DNA (the efficiency can vary depending on the cell type). Therefore, a dominant, selectable marker is used to permit isolation of stable transfectants. In the first part of this unit, the procedure for determining selection conditions and the resulting stable transfection is presented and the most commonly used selectable markers are discussed. The second protocol includes conditions for thirteen markers commonly used for selection of mammalian cells. A third protocols describes selection of transfected cells from the total population soon after transfection with plasmids that express both the gene of interest and a selection tag. Optimization of transfection conditions can be facilitated by a simple staining assay detailed in a .

Purification of Functional RNA‐Protein Complexes using MS2‐MBP

Abstract: Biological machines composed of RNAs and proteins play essential roles in many biological processes. To better understand the mechanism and function of these machines, it is critical to isolate them in a highly purified and functional form. A method for isolating functional RNA-protein complexes assembled in vitro is described. The approach combines gel filtration and an affinity-chromatography strategy using the bacteriophage MS2 coat protein, which binds to a specific RNA-hairpin structure. Using this method, highly purified and functional human spliceosomes have been isolated. The purified spliceosome preparation is used to determine the protein components of the spliceosome by mass spectrometry and to examine the structure of the spliceosome by electron microscopy.

Overview of image analysis, image importing, and image processing using freeware.

Abstract: Quantitative image analysis turns microscopic data that might otherwise be merely descriptive into reliable mechanistic information. This unit provides an overview of the types of analysis that have been useful in the past, as well as descriptions of emerging applications. In addition, two protocols are included for importing data and for the first steps of data manipulation in the more common analytical applications of the freeware, NIH Image and ImageJ, as well as commercially available imaging software, Adobe Photoshop.

Preparation of a Specific Retrovirus Producer Cell Line

Abstract: Establishing a cell line that produces high levels of a specific retrovirus construct involves several steps. First, the retroviral construct must be stably introduced into an appropriate packaging cell line. This can be accomplished either directly by transfection or by transfection to produce a transient virus stock followed by cross-infection of this stock into a separate packaging line; procedures for both approaches are described in this unit. After stable lines are produced, they must be characterized to identify lines that produce high titers of virus with an appropriate structure. A drug selection protocol (the most common method for determining virus titer) is provided along with a sample calculation of BAG virus titer. Sometimes, a short and direct method of estimating virus titer is available when the virus encodes a histochemically detectable gene such as lacZ. For this case, an Xgal staining protocol is described. Similarly, a protocol is provided to estimate the levels of virus being produced by a given cell line by quantitating any specific product of the virus (e.g., RNA) present within the producer line.

Growth and manipulation of S. pombe.

Abstract: This unit presents aspects specific to genetic and cytological manipulation of fission yeast, including mating type testing, crosses, preparingmaking diploids, and analysis of meiotic products, and basic methods of cell cycle synchronization and analysis These methods are different from those used in budding yeast because they depend upon the distinct biology of S pombe, particularly its unwillingness to be a diploid in normal growth conditions Similarly, the different cell shape and growth behavior of S pombe require different approaches to basic cell cycle analysis

Identification of RNA Binding Proteins by UV Cross‐Linking

Abstract: One of the major focuses of modern biology is to understand the dynamics of RNA-protein interactions, including factors that interact with mRNA, rRNA, tRNA, snRNA, hnRNA, siRNA, and viral RNA. Identification the direct interactions between proteins and RNA has greatly advanced our knowledge about the function of RNA-protein machines. UV crosslinking is a straightforward and powerful tool in this endeavor.

Gene Silencing by RNAi in Mammalian Cells

Abstract: This unit provides information how to use short interfering RNA (siRNA) for sequence-specific gene silencing in mammalian cells. Several methods for siRNA generation and optimization, as well as recommendations for cell transfection and transduction, are presented.

Overview of RNA interference and related processes.

Abstract: The history of RNA interference (RNAi) has unfolded rapidly since 1997 with a series of discoveries from plants, fungi, and animals. Initially, the interest was directed towards development of gene silencing technology that could be applied in research, medical therapy, and crop improvement. However, as the underlying mechanism was revealed, it became apparent that RNAi manifests a novel system of genetic regulation that was only hinted at by previous data. This overview unit gives a brief history of the field, describes the natural role of RNAi and related processes, identifies strategies for RNAi and cosuppression in plants and animals, and describes methods for detection and characterization of siRNA and miRNA.

S. pombe Strain Maintenance and Media

Abstract: Fission yeast can be grown and maintained using similar culture methods to those employed for budding yeast. The optimal media for S. pombe is somewhat different than that employed for S. cerevisiae, although budding yeast media can be used if necessary. Good sterile technique is essential to prevent contamination by airborne molds or bacteria.

Detection of Proteins on Blot Transfer Membranes

Abstract: In the basic and alternate protocols of this unit, proteins are stained after electroblotting from polyacrylamide gels to blot transfer membranes. If the samples of interest are electrophoresed in duplicate and transferred to a blot transfer membrane, half of the membrane can be stained to determine the efficiency of transfer to the membrane and the other half can be used for immunoblotting (i.e., western blotting). Detection limits of each staining method are given along with a list of compatible blot transfer membranes and gels. A support protocol describes a method for alkali treatment that enhances subsequent staining of bound proteins.

Introduction of DNA into S. pombe cells.

Abstract: Methods of transformation rely upon conditioning cells to take up DNA, and growing them under selective conditions to establish and maintain the plasmid or integration. Different methods may be used, including electroporation, treatment with lithium cations, or protoplast treatment which removes the cell wall. Protoplasts prepared as for transformation can also be induced to fuse with each other and undergo karyogamy, which provides a means of mating sterile strains.

Overview of Schizosaccharomyces pombe

Abstract: The fission yeast S. pombe provides an attractive alternative system to budding yeast S. cerevisiae for studies of fundamental cell biology. Fission yeast is a particularly powerful model for studies of cell cycle, chromosome dynamics, and polarity. Other areas of study are also expanding. Conceptually similar genetic tools are available in both systems, and both organisms have completely sequenced genomes.

Isotopic assays for reporter gene activity.

Abstract: This unit describes two widely used reporter systems that are based on radioactive detection assays. The first assay uses chloramphenicol acetyltransferase (CAT) activity as a measure of the level of expression of a transfected gene. This bacterial enzyme catalyzes the transfer of an acyl group from acetyl CoA (or any of several other acyl CoA cofactors) to chloramphenicol. In the assays described here, transfected cells are harvested and lysed, and then acyl CoA and radioactively labeled chloramphenicol are added to cell lysate, and modified derivatives of the antibiotic are separated from the starting material using either thin-layer chromatography or phase-extraction. The second reporter system uses a kit to perform a simple two-site radioimmunoassay to quantitate the amount of human growth hormone (hGH) secreted into culture medium by transfected cells. Medium is incubated with 125I-labeled antibody specific for hGH, and immune complexes are collected by an avidin-coated bead. The quantity of hormone is determined based on comparison with a standard curve.

Human Somatic Cell Gene Targeting

Abstract: Human somatic cell gene targeting provides a powerful tool to scientists studying gene function in cultured human cells This technology allows scientists to knock out genes in human somatic cells in a fashion analogous to the creation of knockout mice Human somatic cell gene targeting brings the power of genetics to the study of human genes in human cells by making it possible to compare cells or individuals that are genetically identical except for a single, well-defined mutation in an endogenous gene These modified cells can be studied both in vitro and in vivo This unit presents protocols for human somatic cell gene targeting

Gene expression analysis of a single or few cells

Abstract: The need to analyze rare cells is based on the nature of tissue differentiation and regeneration, the initiation and propagation of disease processes in multicellular organisms, and the functional diversity of individual cells. Gene transcription is the most important regulatory mechanism by which a phenotype and functional state of a cell is determined. Therefore, procedures for the qualitative and quantitative assessment of mRNA abundance are important. This unit presents a protocol for semi-quantitative analysis of gene expression of a single cell and quantitative representation of expressed genes from >10 to 30 cells. A basic protocol for array hybridization on nylon filters is provided because such filters are available in every laboratory. Tissue samples contain many different cell types in variable amounts, so their analysis may require microdissection; a protocol for obtaining cryosections is given. Finally, a simple procedure to prepare the data for statistical analysis is also provided.

Agarose Gel Separation/Isolation of RNA‐Protein Complexes

Abstract: In many methods currently used to analyze RNA-protein complexes, high salt or other stringent treatments are required for reducing nonspecific interactions and resolving the complex of interest. RNA-protein complexes often dissociate on native polyacrylamide gels and can only be detected on density gradients or by gel filtration. Agarose gel electrophoresis provides an alternative method that is simple, rapid, and can have high resolution of RNA-protein complexes. Moreover, the use of low-melting point agarose for the fractionation readily allows for the isolation of the RNA species in each complex detected on the native gel.

Use of monoclonal antibodies for expression cloning.

Abstract: This unit details the use of transient expression in mammalian cells to screen cDNA libraries with monoclonal antibodies (MAb) to isolate cDNA clones encoding cell-surface and intracellular proteins. The first protocol in this unit describes the cloning of cDNAs encoding cell-surface antigens. Several steps in this protocol involve transfection procedures that are described in greater detail elsewhere in this volume. The second protocol is a modification that facilitates isolation of cDNAs encoding antigens that are expressed intracellularly. Both protocols are designed for use with the expression vector CDM8, which contains a polylinker for subcloning double-stranded cDNA.