It is possible to generate tetraploidy in mammalian embryos by chemical or physical suppression of a cleavage division causing endoreduplication of the genome (1,2), or by using techniques to fuse karyoblasts or cells with nucleated or enucleated eggs and blastomeres, which results in tr ...
Electrofusion is a process by which fusion between cell membranes can be induced by exposure to electrical fields (1,2). Many practical applications of electrofusion have been demonstrated, such as the formation of hybridomas (3–6), the production of monoclonal antibodies (MAb) (7–9), s ...
Cell-cell electrofusion (CCE) is a process that involves forcing cells into close juxtaposition and then inducing fusion by delivering electric pulses to the cells. CCE has proven to have many practical applications. It has been used for monoclonal antibody (MAb) production (1,2), hybrid ...
In vitro selection experiments have various goals depending on the composition of the initial pool and the selection method applied. We developed an in vitro selection variant that is useful for the identification of minimal RNA binding sites for proteins within large RNAs. A pool of randomly f ...
Results obtained from in vitro experiments often need to be confirmed by in vivo experiments. The study of RNA-protein interactions is no exception. Information on RNA-protein complex formation in the cell is important for understanding the mechanisms of cellular RNA metabolism such as R ...
Ribonuclease P (RNase P) from Escherichia coli is a transfer RNA (tRNA)-processing enzyme and consists of a catalytic RNA subunit (M1 RNA) and a protein component (C5 protein). M1GS, a gene-targeting ribozyme derived from M1 RNA, can cleave a target messenger RNA (mRNA) efficiently in vitro and inh ...
Many important cellular processes are mediated by sequence-specific RNA binding proteins, and it is often necessary to purify these proteins. When the RNA binding site is known, it is convenient to use this RNA as a matrix for affinity purification. The intronic splicing silencer (ISS) element ...
The understanding of physiology and pathology requires accurate quantification of intracellular concentrations of important molecules such as unique RNA species. Accurate quantification of highly homologous messenger RNAs (mRNAs) (1–3), alternatively spliced mRNAs (4 ...
RNA helicases are essential for the adenosine 5′-triphosphate (ATP)-driven rearrangement of many RNAs and RNA-protein complexes (ribonucleoproteins, RNPs) throughout RNA metabolism. We describe assays to measure RNA and RNP remodeling by RNA helicases in vitro. We show how to prepa ...
Terminal transferase-dependent polymerase chain reaction (TDPCR) can be used after reverse transcription (RT) to analyze RNA. This method (RT-TDPCR) has been used for study of RNA structure and RNA-protein interactions at nucleotide-level resolution. A detailed protocol of RT-TD ...
Small interfering RNA (siRNA)-mediated RNA interference (RNAi) is a very powerful tool for triggering posttranscriptional gene silencing in several organisms. We discuss the improvement of two different sources of siRNAs synthesized either chemically or by an enzymatic method. ...
Pre-mRNA (messenger RNA) splicing is an essential step for gene expression in higher eukaryotes. Splicing reactions have been well studied in vitro using extracts prepared from cultured cells. We describe protocols for the preparation of splicing-competent extracts from whole cel ...
Biochemical assay of proteomic libraries derived from the Saccharomyces cerevisiae genome provides a powerful new tool for the assignment of activities to proteins. Particular advantages of this approach include the speed with which a protein can be identified and the generality for ...
Antisense oligonucleotides have been used to study the structure and function of small nuclear ribonucleoprotein (snRNP) complexes and were adapted and modified for the purification of a variety of RNPs. We describe methods for recombinant expression and reconstitution of cataly ...
Isolation of ribonucleoprotein particles from living cells and cell lysates has allowed the identification of both simple bimolecular interactions and the members of large, extended complexes. A number of different strategies have been devised to isolate these complexes by using a ...
Studies of RNA—protein interactions often require assembly of the RNA—protein complex using in vitro synthesized RNA or recombinant protein. Here, we describe a protocol to assemble a functional spliceosome in yeast extracts using transcribed or synthetic RNAs. The in vitro assembl ...
Recently developed affinity purification methods have revolutionized our understanding of the higher-ordered structures of multisubunit, often low-abundance macromolecular complexes, including ribonucleoproteins (RNPs). Often, purification by classical, no ...
We present a newly developed method for fixing RNA-protein complexes in situ in living cells and the subsequent purification of the RNA targets. Using this approach, complex tissue such as mouse brain can be ultraviolet (UV) irradiated to covalently crosslink RNA-protein complexes. Once c ...
Isothermal titration calorimetry (ITC) is a useful technique to study RNA-protein interactions as it provides the only method by which the thermodynamic parameters of free energy, enthalpy, and entropy can be directly determined. This chapter presents a general procedure for studyi ...
The gel mobility shift assay is routinely used to visualize protein—RNA interactions. Its power resides in the ability to resolve free from bound RNA with high resolution in a gel matrix. We review the quantitative application of this approach to elucidate thermodynamic properties of prot ...
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