Springer protocols feed by Genetics/Genomics 
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This is an RSS file. You can use it to subscribe to this data in your favourite RSS reader or to display this data on your own website or blog.Therapeutic Genome Mutagenesis Using Synthetic Donor DNA and Triplex-Forming Molecules
Genome mutagenesis can be achieved in a variety of ways, though a select few are suitable for therapeutic settings. Among them, the harnessing of intracellular homologous recombination affords the safety and efficacy profile suitable for such settings. Recombinagenic donor DNA and mutagenic triplex-forming molecules co-opt this natural recombination phenomenon to enable the specific, heritable editing and targeting of the genome. Editing the genome is achieved by designing the sequence-specific recombinagenic donor DNA to have base mismatches, insertions, and deletions that will be incorporated into the genome when it is u...
Source: Springer protocols feed by Genetics/Genomics - December 22, 2014 Category: Genetics & Stem Cells Source Type: news
Using Phage Integrases in a Site-Specific Dual Integrase Cassette Exchange Strategy
ΦC31 integrase, a site-specific large serine recombinase, is a useful tool for genome engineering in a variety of eukaryotic species and cell types. ΦC31 integrase performs efficient recombination between its attB site and either its own placed attP site or a partially mismatched genomic pseudo attP site. Bxb1 integrase, another large serine recombinase, has a similar level of recombinational activity, but recognizes only its own attB and attP sites. Previously, we have used these integrases sequentially to integrate plasmid DNA into the genome. This approach relied on placing a landing pad attP for Bxb1 integrase ...
Source: Springer protocols feed by Genetics/Genomics - December 22, 2014 Category: Genetics & Stem Cells Source Type: news
piggyBac Transposon-Based Insertional Mutagenesis in Mouse Haploid Embryonic Stem Cells
Forward genetic screening is a powerful non-hypothesis-driven approach to unveil the molecular mechanisms and pathways underlying phenotypes of interest. In this approach, a genome-wide mutant library is first generated and then screened for a phenotype of interest. Subsequently, genes responsible for the phenotype are identified. There have been a number of successful screens in yeasts, Caenorhabditis elegans and Drosophila. These model organisms all allow loss-of-function mutants to be generated easily on a genome-wide scale: yeasts have a haploid stage in their reproductive cycles and the latter two organisms have short...
Source: Springer protocols feed by Genetics/Genomics - December 22, 2014 Category: Genetics & Stem Cells Source Type: news
Genome Editing by Targeted Chromosomal Mutagenesis
The tools for genome engineering have become very powerful and accessible over the last several years. CRISPR/Cas nucleases, TALENs and ZFNs can all be designed to produce highly specific double-strand breaks in chromosomal DNA. These breaks are processed by cellular DNA repair machinery leading to localized mutations and to intentional sequence replacements. Because these repair processes are common to essentially all organisms, the targetable nucleases have been applied successfully to a wide range of animals, plants, and cultured cells. In each case, the mode of delivery of the nuclease, the efficiency of cleavage and t...
Source: Springer protocols feed by Genetics/Genomics - November 20, 2014 Category: Genetics & Stem Cells Source Type: news
Simple Sperm Preservation by Freeze-Drying for Conserving Animal Strains
Freeze-drying spermatozoa is the ultimate method for the maintenance of animal strains, in that the gametes can be preserved for a long time in a refrigerator at 4 °C. Furthermore, it is possible to realize easy and safe transportation of spermatozoa at an ambient temperature that requires neither liquid nitrogen nor dry ice. Freeze-drying spermatozoa has been established as a new method for storing genetic resources instead of cryopreservation using liquid nitrogen. This chapter introduces our latest protocols for freeze-drying of mouse and rat spermatozoa, and the anticipated results of the fertilizing ability of the...
Source: Springer protocols feed by Genetics/Genomics - November 20, 2014 Category: Genetics & Stem Cells Source Type: news
Creating Knockout and Knockin Rodents Using Engineered Endonucleases via Direct Embryo Injection
Genetically engineered rodents have been generated worldwide for biomedical research. Recently, gene targeting techniques have been developed by using engineered endonucleases such as zinc-finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN) and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9. These endonucleases are useful for simple and rapid production of gene knockout/knockin animals without using embryonic stem (ES) cells. This chapter introduces the latest protocols for producing genetically modified rodents using ZFN, TALEN, and CRISPR/Cas9. (Source: Springer protoc...
Source: Springer protocols feed by Genetics/Genomics - November 20, 2014 Category: Genetics & Stem Cells Source Type: news
Using Engineered Endonucleases to Create Knockout and Knockin Zebrafish Models
Over the last few years, the technology to create targeted knockout and knockin zebrafish animals has exploded. We have gained the ability to create targeted knockouts through the use of zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats/CRISPR associated system (CRISPR/Cas). Furthermore, using the high-efficiency TALEN system, we were able to create knockin zebrafish using a single-stranded DNA (ssDNA) protocol described here. Through the use of these technologies, the zebrafish has become a valuable vertebrate model and an e...
Source: Springer protocols feed by Genetics/Genomics - November 20, 2014 Category: Genetics & Stem Cells Source Type: news
Strategies to Increase Genome Editing Frequencies and to Facilitate the Identification of Edited Cells
The power of genome editing is increasingly recognized as it has become more accessible to a wide range of scientists and a wider range of uses has been reported. Nonetheless, an important practical aspect of the strategy is develop methods to increase the frequency of genome editing or methods that enrich for genome-edited cells such that they can be more easily identified. This chapter discusses several different approaches including the use of cold-shock, exonucleases, surrogate markers, specialized donor vectors, and oligonucleotides to enhance the frequency of genome editing or to facilitate the identification of geno...
Source: Springer protocols feed by Genetics/Genomics - November 20, 2014 Category: Genetics & Stem Cells Source Type: news
Genome Editing in Human Pluripotent Stem Cells Using Site-Specific Nucleases
Human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) (Thomson, Science 282:1145–1147, 1998; Takahashi et al. Cell 131:861–872, 2007), collectively referred to as pluripotent stem cells (hPSCs), are currently used in disease modeling to address questions specific to humans and to complement our insight gained from model organisms (Soldner et al. Cell 146:318–331, 2011; Soldner and Jaenisch, Science 338:1155–1156, 2012). Recently, genetic engineering using site-specific nucleases has been established in hPSCs (Hockemeyer et al. Nat Biotechnol 27:851–857, 2009; Hockemeyer...
Source: Springer protocols feed by Genetics/Genomics - November 20, 2014 Category: Genetics & Stem Cells Source Type: news
Gene Editing Using ssODNs with Engineered Endonucleases
Gene editing using engineered endonucleases, such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 nucleases, requires the creation of a targeted, chromosomal DNA double-stranded break (DSB). In mammalian cells, these DSBs are typically repaired by one of the two major DNA repair pathways: nonhomologous end joining (NHEJ) or homology-directed repair (HDR). NHEJ is an error-prone repair process that can result in a wide range of end-joining events that leads to somewhat random mutations at the site of DSB. H...
Source: Springer protocols feed by Genetics/Genomics - November 20, 2014 Category: Genetics & Stem Cells Source Type: news
Silencing Long Noncoding RNAs with Genome-Editing Tools
Long noncoding RNAs (lncRNAs) are a functional and structural diverse class of cellular transcripts that comprise the largest fraction of the human transcriptome. However, detailed functional analysis lags behind their rapid discovery. This might be partially due to the lack of loss-of-function approaches that efficiently reduce the expression of these transcripts. Here, I describe a method that allows a specific and efficient targeting of the highly abundant lncRNA MALAT1 in human (lung) cancer cells. The method relies on the site-specific integration of RNA-destabilizing elements mediated by Zinc Finger Nucleases (ZFNs)....
Source: Springer protocols feed by Genetics/Genomics - November 20, 2014 Category: Genetics & Stem Cells Source Type: news
Endogenous Gene Tagging with Fluorescent Proteins
Human genome manipulation has become a powerful tool for understanding the mechanisms of numerous diseases including cancer. Inserting reporter sequences in the desired locations in the genome of a cell can allow monitoring of endogenous activities of disease related genes. Native gene expression and regulation is preserved in these knock-in cells in contrast to cell lines with target overexpression under an exogenous promoter as in the case of transient transfection or stable cell lines with random integration. The fusion proteins created using the modern genome editing tools are expressed at their physiological level and...
Source: Springer protocols feed by Genetics/Genomics - November 20, 2014 Category: Genetics & Stem Cells Source Type: news
Donor Plasmid Design for Codon and Single Base Genome Editing Using Zinc Finger Nucleases
In recent years, CompoZr zinc finger nuclease (ZFN) technology has matured to the point that a user-defined double strand break (DSB) can be placed at virtually any location in the human genome within 50 bp of a desired site. Such high resolution ZFN engineering is well within the conversion tract limitations demarcated by the mammalian DNA repair machinery, resulting in a nearly universal ability to create point mutations throughout the human genome. Additionally, new architectures for targeted nuclease engineering have been rapidly developed, namely transcription activator like effector nucleases (TALENs) and clustered r...
Source: Springer protocols feed by Genetics/Genomics - November 20, 2014 Category: Genetics & Stem Cells Source Type: news
Genome Engineering Using CRISPR-Cas9 System
We present all relevant methods including the initial site selection, molecular cloning, delivery of guide RNAs (gRNAs) and Cas9 into mammalian cells, verification of target cleavage, and assays for detecting genomic modification including indels and homologous recombination. These tools provide researchers with new instruments that accelerate both forward and reverse genetics efforts. (Source: Springer protocols feed by Genetics/Genomics)
Source: Springer protocols feed by Genetics/Genomics - November 20, 2014 Category: Genetics & Stem Cells Source Type: news
Assessing Intermolecular RNA:RNA Interactions Within a Ribonucleoprotein Complex Using Heavy Metal Cleavage Mapping
Heavy metal cleavage mapping analysis of both assembling and fully mature ribonucleoprotein (RNP) complexes are informative techniques for assessing the intermolecular base pairing between small non-coding RNAs and their interacting target RNAs. Lead cleavage of the RNA in partially or fully assembled RNPs in the absence or presence of the interacting RNA can determine both the accessibility of the base pairing sequence within the RNP itself as well as its interaction with the target RNA. In this chapter, we detail how this technique was used to map the intermolecular RNA:RNA base pairing of a box C/D RNA with its target R...
Source: Springer protocols feed by Genetics/Genomics - October 30, 2014 Category: Genetics & Stem Cells Source Type: news
