Titlebook: In Vitro Mutagenesis; Methods and Protocol Andrew Reeves Book 2017 Springer Science+Business Media New York 2017 gene and genome editing.fa

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CRISPR/Cas9-Mediated Mutagenesis of Human Pluripotent Stem Cells in Defined Xeno-Free E8 Medium (hPSCs). In addition to facilitating hPSC-based disease studies, the application of genome engineering in hPSCs has also opened up new avenues for cell replacement therapy. To improve consistency and reproducibility of hPSC-based studies, and to meet the safety and regulatory requirements for clini

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Development of CRISPR/Cas9 for Efficient Genome Editing in ,ears, the RNA-guided Cas9 nucleases derived from the prokaryotic type 2 CRISPR (clustered regularly interspaced short palindromic repeats) systems have drastically improved our ability to engineer the genomes of a variety of organisms including .. In this chapter, we describe detailed protocols for

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Generation of Stable Knockout Mammalian Cells by TALEN-Mediated Locus-Specific Gene Editingely effective and specific knockout strategy in both cultured cells and animal models. The current chapter describes a protocol for the construction and generation of TALENs using serial and hierarchical digestion and ligation steps, and using the synthesized TALEN pairs to achieve locus-specific ta

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Efficient Generation of Gene-Modified Mice by Haploid Embryonic Stem Cell-Mediated Semi-cloned Technle pronucleus from zygotes. These cells, termed AG-haESCs, can be used in place of sperm to produce the so-called semi-cloned (SC) mice. Importantly, AG-haESCs carrying .-DMR and .-DMR knockouts (DKO-AG-haESCs) can efficiently and stably support the generation of SC mice via intracytoplasmic AG-haES

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Insertion of Group II Intron-Based Ribozyme Switches into Homing Endonuclease Genesve tools for genome editing, targeted mutagenesis and gene therapy applications. Herein, we present strategies where homing endonuclease open reading frames (HEases ORFs) are interrupted with group II intron sequences. The ultimate goal is to achieve in vivo expression of HEases that can be regulate

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Generating a Genome Editing Nuclease for Targeted Mutagenesis in Human Cells invented to fulfill these purposes, including zinc finger nucleases, TALEN, and CRISPR/Cas9. Although all of these systems can target DNA sequence with high efficiency, they also exert off-target effects and genotoxicity. The off-target effects might not hinder their usage in animal models because

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Use of Group II Intron Technology for Targeted Mutagenesis in ,trategies for bacterial pathogens. This chapter will discuss the application of a targeted, intron-based insertional mutagenesis method for creating mutants in the obligate, intracellular bacterial pathogen .. The methods employed for intron targeting, mutant selection, and mutant verification will

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In Silico Approaches to Identify Mutagenesis Targets to Probe and Alter Protein–Cofactor and Proteinrotein under study. It is also very useful to have structures of multiple related proteins in order to determine whether or not particular amino acid residues are conserved in the structures either in the active site of an enzyme at the surface of a protein or at a putative protein–protein interface

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