遗传学实验技术

Current baculovirus expression vector systems (BEVS) rely on either using homologous recombination or site specific transposition (Tn7 transposition) to obtain recombinant baculovirus. Each approach has its own merits. To date, the choice of transfer plasmids limited expres ...

This chapter describes the step-by-step methods employed by the Structural Genomics Consortium (SGC) for screening and producing proteins in the baculovirus expression vector system (BEVS). This eukaryotic expression system was selected and a screening process established in ...

In Chapter 4 we described the SGC process for generating multiple constructs of truncated versions of each protein using LIC. In this chapter we provide a step-by-step procedure of our E. coli system for test expressing intracellular (soluble) proteins in a 96-well format that enables us to ident ...

Structural genomics groups have identified the need to generate multiple truncated versions of each target to improve their success in producing a well-expressed, soluble, and stable protein and one that crystallizes and diffracts to a sufficient resolution for structural determ ...

The protocols outlined in this chapter allow for the small-scale test expression of a single or multiple proteins concurrently using several expression conditions to identify optimal strategies for producing soluble, stable proteins. The protocols can be performed manually wit ...

Inducible production of proteins from cloned genes in E. coli is widely used, economical, and effective. However, common practices can result in unintended induction, inadvertently generating cultures that give poor or variable yields in protein production. Recipes are provided for ...

Intrinsically disordered or unstructured regions in proteins are both common and biologically important, particularly in regulation, signaling, and modulating intermolecular recognition processes. From a practical point of view, however, such disordered regions often c ...

When eukaryotic proteins are overexpressed in Escherichia coli hosts, they often form inclusion bodies. Natively folded proteins can be extracted from inclusion bodies using mild detergents such as sarkosyl. One common problem is the sequestration of nucleic acid contaminants wi ...

Recent advances in cell-free protein expression systems have made them reliable and practical for functional and structural studies of a wide variety of proteins. In particular, wheat germ cell-free translation can consistently produce target proteins in microgram quantities fr ...

Cell-free protein synthesis is advantageous for the expression of protein complexes, since it is suitable for the co-expression of two or more components of the target protein complexes. The quantity and the quality of cell-free expressed complexes are generally better than those of prot ...

The ever-increasing number of different miRNAs and their association with a vast number of cellular dysfunctions and diseases have initiated several groups to investigate miRNA maturation, which ultimately leads to down regulation of a target messenger RNA (mRNA) and its downstream ...

We used fluorescence correlation spectroscopy (FCS) to establish an in vitro assay to investigate RNase activity of human Dicer (Werner et al., Biol Chem 393(3):187–193). FCS allows investigating the interactions of different particles due to their differing diffusion mobility, prov ...

Assaying Dicer-mediated miRNA maturation is a valuable tool not only for validating miRNA maturation itself but also for testing Dicer activity in cell lysate and for screening small molecules inhibiting miRNA maturation in a high-throughput format. The classical assay for miRNA mat ...

Luciferase reporter assays are widely used to study promoter activity, transcription factors, intracellular signaling, protein interactions (Jia et al., PloS One 6:e26414), miRNA processing (Allegra and Mertens, Biochem Biophys Res Commun 406:501–505), and target recognition ...

In animals, the Microprocessor complex cleaves primary transcripts of microRNAs (pri-miRNAs) to produce precursor microRNAs in the nucleus. The core components of Microprocessor include the Drosha ribonuclease and its RNA-binding partner protein DiGeorge critical region 8 ( ...

The quantitative real-time polymerase chain reaction (qRT-PCR) is a valuable and well-proven technique used to investigate the expression level of multiple components of the microRNA (miRNA) maturation machinery. Here, we describe how to determine the messenger RNA expression le ...

Deregulation of microRNAs (miRNAs) has been attributed to almost any human disease analyzed to date. This calls for models and experimental strategies for functional analyses of miRNAs enabling miRNA overexpression or suppression in target cell and/or tissues. Lentiviral vector ( ...

The involvement of microRNAs in human pathologies is a firmly established fact. Accordingly, the pharmacological modulation of their activity appears to be a very appealing issue in the development of new types of drugs (miRNA therapeutics). One of the most interesting issues is the possib ...

Molecules able to interfere in miRNA genesis and function are potent tools to unravel maturation and processing pathways. Antisense oligonucleotides or analogs are actually employed for the inhibition of miRNA function. Here we illustrate how Peptide Nucleic Acids oligomers targ ...

Aberrant expression of microRNAs (miRNAs) has been linked to many human diseases including cancer, immune disorders, heart disease, and viral infections. Thus, small molecule inhibitors of miRNAs have potential as new therapeutic agents, as probes for the elucidation of detailed mec ...