Methods in molecular biology
-
The machine learning field, which can be briefly defined as enabling computers make successful predictions using past experiences, has exhibited an impressive development recently with the help of the rapid increase in the storage capacity and processing power of computers. Together with many other disciplines, machine learning methods have been widely employed in bioinformatics. ⋯ Then, we point out the main issues of designing machine learning experiments and their performance evaluation. Finally, we introduce some supervised learning methods.
-
The clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) systems have evolved as an adaptive surveillance and defense mechanism in bacteria and archaea that uses short RNAs to direct degradation of foreign genetic elements. Here, we present our protocol for utilizing the S. pyogenes type II bacterial CRISPR system to achieve sequence-specific genome alterations in human cells. ⋯ Genomic alterations at the target site are then introduced either through nonhomologous end joining (NHEJ) or through homologous recombination (HR) in the presence of an appropriate donor sequence. This RNA-guided editing tool offers greater ease of customization and synthesis in comparison to existing sequence-specific endonucleases and promises to become a highly versatile and multiplexable human genome engineering platform.
-
Blindness is one of the most devastating conditions affecting the quality of life. Hereditary degenerative diseases, such as retinitis pigmentosa, are characterized by the progressive loss of photoreceptors, leading to complete blindness. No treatment is known, the current state-of-the-art of restoring vision are implanted electrode arrays. ⋯ Successful treatment strategies have to take into account this diversity, as only the existing retinal hardware can serve as substrate for optogenetic intervention. The goal is to salvage the retinal ruins and to revert the leftover tissue into a functional visual sensor that operates as optimally as possible. Here, we discuss three different successful approaches that have been applied to degenerated mouse retina.
-
iRefWeb is a bioinformatics resource that offers access to a large collection of data on protein-protein interactions in over a thousand organisms. This collection is consolidated from 14 major public databases that curate the scientific literature. ⋯ Users of iRefWeb are able to retrieve all curated interactions for a given organism or those involving a given protein (or a list of proteins), narrow down their search results based on different supporting evidence, and assess the reliability of these interactions using various criteria. They may also examine all data and annotations related to any publication that described the interaction-detection experiments. iRefWeb is freely available to the research community worldwide at http://wodaklab.org/iRefWeb .
-
Stem cells are envisaged to be integral components of multicellular systems engineered for therapeutic applications. The reprogramming of somatic cells to induced pluripotent stem cells (iPSCs) via recombinant expression of a limited number of transcription factors, which was first achieved by Yamanaka and colleagues in 2007, heralded a major breakthrough in the stem cell field. Since then, there has been rapid progress in the field of iPSC generation, including the identification of various small molecules that can enhance reprogramming efficiency and reduce the number of different transcription factors required for reprogramming. ⋯ The use of recombinant cell-penetrating peptides and direct transfection of synthetic mRNA encoding appropriate transcription factors have both been shown to successfully reprogram somatic cells to iPSCs. It has also been shown more recently that the direct transfection of certain miRNA species can reprogram somatic cells to pluripotency without the need for any of the transcription factors commonly utilized for iPSC generation. This chapter describes protocols for iPSC generation with these new techniques, which would obviate the use of recombinant DNA and viral vectors in cellular reprogramming, thus avoiding permanent genetic modification to the reprogrammed cells.