Download or read book Gene Targeting Protocols written by Eric B. Kmiec and published by Humana. This book was released on 2013-08-14 with total page 233 pages. Available in PDF, EPUB and Kindle. Book excerpt: The potential now exists in many experimental systems to transfer a cloned, modified gene back into the genome of the host organism. In the ideal situation, the cloned gene is returned to its homologous location in the genome and becomes inserted at the target locus. This process is a controlled means for the repair of DNA damage and ensures accurate chromosome disjunction during meiosis. The paradigm for thinking about the mechanism of this p- cess has emerged primarily from two sources: (1) The principles of reaction mechanics have come from detailed biochemical analyses of the RecA protein purified from Escherichia coli; and (2) the principles of information transfer have been derived from genetic studies carried out in bacteriophage and fungi. A compelling picture of the process of homologous pairing and DNA strand exchange has been influential in directing investigators interested in gene t- geting experiments. The ability to find and pair homologous DNA molecules enables ac- rate gene targeting and is the central phenomenon underlying genetic recombi- tion. Biochemically, the overall process can be thought of as a series of steps in a reaction pathway whereby DNA molecules are brought into homologous register, the four-stranded Holliday structure intermediate is formed, hete- duplex DNA is extended, and DNA strands are exchanged. Not much is known about the biochemical pathway leading to homologous recombination in euka- otes.
Download or read book Gene Knockout Protocols written by Martin J. Tymms and published by Springer Science & Business Media. This book was released on 2008-02-03 with total page 434 pages. Available in PDF, EPUB and Kindle. Book excerpt: As the major task of sequencing the human genome is near completion and full complement of human genes are catalogued, attention will be focused on the ultimate goal: to understand the normal biological functions of these genes, and how alterations lead to disease states. In this task there is a severe limitation in working with human material, but the mouse has been adopted as the favored animal model because of the available genetic resources and the highly conserved gene conservation linkage organization. In just of ten years since the first gene-targeting experiments were p- formed in embryonic stem (ES) cells and mutations transmitted through the mouse germline, more than a thousand mouse strains have been created. These achievements have been made possible by pioneering work that showed that ES cells derived from preimplantation mouse embryos could be cultured for prolonged periods without differentiation in culture, and that homologous rec- bination between targeting constructs and endogenous DNA occurred at a f- quency sufficient for recombinants to be isolated. In the next few years the mouse genome will be systematically altered, and the techniques for achi- ing manipulations are constantly being streamlined and improved.
Download or read book Laboratory Protocols for Conditional Gene Targeting written by Raul M. Torres and published by . This book was released on 2004 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt:
Download or read book Gene Targeting written by Alexandra Joyner and published by OUP Oxford. This book was released on 1999-12-09 with total page 312 pages. Available in PDF, EPUB and Kindle. Book excerpt: Since the publication of the first edition of Gene Targeting: A Practical Approach in 1993 there have been many advances in gene targeting and this new edition has been thoroughly updated and rewritten to include all the major new techniques. It provides not only tried-and-tested practical protocols but detailed guidance on their use and applications. As with the previous edition Gene Targeting: A Practical Approach 2e concentrates on gene targeting in mouse ES cells, but the techniques described can be easily adapted to applications in tissue culture including those for human cells. The first chapter covers the design of gene targeting vectors for mammalian cells and describes how to distinguish random integrations from homologous recombination. It is followed by a chapter on extending conventional gene targeting manipulations by using site-specific recombination using the Cre-loxP and Flp-FRT systems to produce 'clean' germline mutations and conditionally (in)activating genes. Chapter 3 describes methods for introducing DNA into ES cells for homologous recombination, selection and screening procedures for identifying and recovering targeted cell clones, and a simple method for establishing new ES cell lines. Chapter 4 discusses the pros and cons or aggregation versus blastocyst injection to create chimeras, focusing on the technical aspects of generating aggregation chimeras and then describes some of the uses of chimeras. The next topic covered is gene trap strategies; the structure, components, design, and modification of GT vectors, the various types of GT screens, and the molecular analysis of GT integrations. The final chapter explains the use of classical genetics in gene targeting and phenotype interpretation to create mutations and elucidate gene functions. Gene Targeting: A Practical Approach 2e will therefore be of great value to all researchers studying gene function.
Download or read book Laboratory Protocols for Conditional Gene Targeting written by Raul M. Torres and published by . This book was released on 1997 with total page 167 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Download or read book Methods in Molecular Biology Gene targeting protocols written by John M. Walker and published by . This book was released on 1984 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt:
Download or read book Transgenic Mouse Methods and Protocols written by Marten H. Hofker and published by Springer Science & Business Media. This book was released on 2008-02-04 with total page 382 pages. Available in PDF, EPUB and Kindle. Book excerpt: Marten Hofker and Jan van Deursen have assembled a multidisciplinary collection of readily reproducible methods for working with mice, and particularlyfor generating mouse models that will enable us to better understand gene function. Described in step-by-step detail by highly experienced investigators, these proven techniques include new methods for conditional, induced knockout, and transgenic mice, as well as for working with mice in such important research areas as immunology, cancer, and atherosclerosis. Such alternative strategies as random mutagenesis and viral gene transduction for studying gene function in the mouse are also presented.
Download or read book Gene Targeting and Embryonic Stem Cells written by Jim Mcwhir and published by Garland Science. This book was released on 2004-03-01 with total page 152 pages. Available in PDF, EPUB and Kindle. Book excerpt: Gene Targeting and Embryonic Stem Cells is a practical guide designed for the rapidly growing number of researchers who are moving into this field. Provides details on how to culture, transfect and differentiate established cell lines, and how to isolate new cell lines. Gene targeting experiments are described for a number of cell types, including ungulate fetal fibroblasts, murine ES cells, human embryonal carinoma cells and human ES cells, and include protocols for gene-targeting vectors, DNA transfection and RNA interference. The recent isolation of human embryonic stem cells and the potential of these cells for therapeutic applications has generated an entirely new methodology. Similarly, gene targeting methodology has recently been extended to nuclear donor cells in ungulate species. This volume will be invaluable for both new and established researchers in the field of human embryonic stem cells, and to biotech companies engaged in the production of transgenic proteins in livestock, xenotransplantation and the development of animal models.
Download or read book Gene Correction written by Francesca Storici and published by Humana. This book was released on 2016-09-03 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: Gene correction is a technology that gives us the tools for both repairing and mutating DNA, for discovering gene functions and for engineering new genetic variants. Gene Correction: Methods and Protocols provides a user friendly, detailed and up-to-date collection of strategies and methodologies utilized for generating specific sequence changes in the DNA of cells in the laboratory, while also tackling the major problems that the field of gene correction faces. This volume brings together many experts in the field of gene correction to disclose a wide and varied array of specific gene correction protocols for engineering mutations in DNA, for delivering correcting DNA to target cells, and for improving the accuracy and safety of the gene correction process. Written in the successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible protocols, and notes on troubleshooting and avoiding known pitfalls. Authoritative and easily accessible, Gene Correction: Methods and Protocols seeks to serve scientists of all backgrounds interested in the area of gene targeting/recombination/therapy.
Download or read book Gene Knockout Protocols written by Ralf Kühn and published by Humana Press. This book was released on 2009-03-27 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: Following the completion of the mouse and human genome sequences, a major challengeisthefunctionalcharacterizationofeverymammaliangeneandthedeciph- ing of their molecular interaction network. The mouse offers many advantages for the use of genetics to study human biology and disease, unmatched among other m- mals. Its development, body plan, physiology, behavior, and diseases have much in common, based on the fact that 99% of the human genes have a mouse ortholog. The investigation of gene function using mouse models is based on many years of tech- logical development. In the two decades since gene targeting in murine embryonic stem (ES) cells was first described by Mario Capecchi and colleagues, more than 3000 predesigned mouse mutants have been developed. To date, a variety of mouse mutagenesis techniques, either gene- or phenotype-driven, are used as systematic approaches. The availability of the genome sequence supports gene-driven approaches such as gene-trap and targeted mutagenesis in ES cells, allowing efficient and precise gene disruption. In combination with the use of site-specific DNA recombinases, in particular the Cre/loxP system, gene disruptioncan be directed to specific cell types in conditionalmousemutants. Furthermore,chemicalandtransposonmutagenesisofthe mouse genome enables us to perform phenotype-driven screens for the unbiased identification of phenotype–genotype correlations involved in models of human d- ease. Over the next several years, the mouse genome will be systematically altered, and the techniques for achieving predesigned manipulations will be constantly developed further and improved. The second edition of Gene Knockout Protocols brings together distinguished c- tributorswithextensiveexperienceinthegenetargetingandmousegeneticsfields.
Download or read book Lentivirus Gene Engineering Protocols written by Maurizio Federico and published by Springer Science & Business Media. This book was released on 2008-02-03 with total page 310 pages. Available in PDF, EPUB and Kindle. Book excerpt: Cell gene engineering is emerging as a field with outstanding impact, not only in medicine/biology, but also, and perhaps most importantly, in agriculture and in all those food sciences involved in the fight against world hunger. Lentivirus vector-based technologies represent the last frontier in the development of powerful and reliable methods for both in vitro and in vivo gene transfer in eukaryotic animal cells. Although the design of lentivirus vectors is closely reminiscent of those already successfully applied to the construction of oncoretroviral vectors, some unique features, e.g., the ef- ciency in transducing both postmitotic and stem cells, render the use of lentivirus vectors invaluable. It has been a great pleasure to edit Lentivirus Gene Engineering Pro- cols, owing in part to the high level of enthusiasm that the authors dem- strated in contributing to this book. The fact that so many outstanding scientists engaged in lentivirus vector research have provided articles renders it so- thing more than a technical handbook. In addition to detailed descriptions of the most innovative methodologies, the reader may find very informative ov- views concerning both theoretical and practical aspects of the origin and the development of diverse lentivirus vector types. This, in my opinion, rep- sents a unique added value of this volume, which should help our work resist the passage of time, to which books such as this are particularly sensitive.
Download or read book Target Discovery and Validation Reviews and Protocols written by Mouldy Sioud and published by Springer Science & Business Media. This book was released on 2008-02-04 with total page 360 pages. Available in PDF, EPUB and Kindle. Book excerpt: Target discovery is a field that has existed for several years but is so vibrant today because of the recent progress in our understanding of the molecular mechanisms of many human diseases and the technical advances in target identification and validation. More sophisticated gene profiling technologies, such as DNA microarrays and serial analysis of gene expression, permit rapid identification of lead targets. Moreover, analysis of gene networks in living organisms allows the identification of target genes that operate in defined physiological pathways. With the sequencing of several genomes completed and the rapidly growing gene expression databases, there is now greater impetus than ever before for in silico discovery of therapeutic targets. Also, recent advances in genetic technologies have increased our ability to generate mouse models for human diseases. The implications of these genetically modified animals in drug development are several, including identification of new drug targets, predicting efficacy, and uncovering possible side effects. Together, these recent technical advances should allow researchers to make the most informed choice early and advance the chosen targets toward clinical studies. Regarding cancers, any difference between a cancer and a normal cell could potentially be exploited as a therapeutic target. The hope is that drugs targeting specific constituents or pathways in cancer cells will provide more effective therapy, either alone or in combination with other currently used anticancer drugs. In addition to drug targets, identifying new target antigens remains as much of a challenge as improving tumor vaccines already in the clinic.
Download or read book Transgenesis Techniques written by Alan R. Clarke and published by Springer Science & Business Media. This book was released on 2008-02-05 with total page 346 pages. Available in PDF, EPUB and Kindle. Book excerpt: The past decade has witnessed a spectacular explosion in both the devel- ment and use of transgenic technologies. Not only have these been used to aid our fundamental understanding of biologic mechanisms, but they have also faci- tated the development of a range of disease models that are now truly beginning to impact upon our approach to human disease. Some of the most exciting model systems relate to neurodegenerative disease and cancer, where the availability of appropriate models is at last allowing radically new therapies to be developed and tested. This latter point is of particular significance given the current concerns of the wider public over both the use of animal models and the merits of using genetically modified organisms. Arguably, advances of the greatest significance have been made using mammalian systems—driven by the advent of embryonic stem-cell–based strategies and, more recently, by cloning through nuclear transfer. For this reason, this new edition of Transgenesis Techniques focuses much more heavily on manipulation of the mammalian genome, both in the general discussions and in the provision of specific protocols.
Download or read book Genetic Recombination written by Alan S. Waldman and published by Springer Science & Business Media. This book was released on 2008-02-03 with total page 259 pages. Available in PDF, EPUB and Kindle. Book excerpt: Genetic recombination, in the broadest sense, can be defined as any process in which DNA sequences interact and undergo a transfer of information, producing new “recombinant” sequences that contain information from each of the original molecules. All organisms have the ability to carry out recombination, and this striking universality speaks to the essential role recombination plays in a variety of biological processes fundamentally important to the maintenance of life. Such processes include DNA repair, regulation of gene expression, disease etiology, meiotic chromosome segregation, and evolution. One important aspect of recombination is that it typically occurs only between sequences that display a high degree of sequence identity. The stringent requirement for homology helps to ensure that, under normal circumstances, a cell is protected from deleterious rearrangements since a swap of genetic information between two nearly identical sequences is not expected to dramatically alter a genome. Recombination between dissimilar sequences, which does happen on occasion, may have such harmful consequences as chromosomal translocations, deletions, or inversions. For many organisms, it is also important that recombination rates are not too high lest the genome become destabilized. Curiously, certain organisms, such as the trypanosome parasite, actually use a high rate of recombination at a particular locus in order to switch antigen expression continually and evade the host immune system effectively.
Download or read book Advanced Protocols for Animal Transgenesis written by Shirley Pease and published by Springer Nature. This book was released on 2011-08-30 with total page 671 pages. Available in PDF, EPUB and Kindle. Book excerpt: This laboratory manual, published in cooperation with the International Society for Transgenic Technology (ISTT), provides almost all current methods that can be applied to the creation and analysis of genetically modified animals. The chapters have been contributed by leading scientists who are actively using the technology in their laboratories. Based on their first-hand experience the authors also provide helpful notes and troubleshooting sections. Topics range from standard techniques, such as pronuclear microinjection of DNA, to more sophisticated and modern methods, such as the derivation and establishment of embryonic stem (ES) cell lines, with defined inhibitors in cell culture medium. In addition, related topics with relevance to the field are addressed, including global web-based resources, legal issues, colony management, shipment of mice and embryos, and the three R’s: refinement, reduction and replacement.
Download or read book Genome Editing in Animals written by Izuho Hatada and published by Springer Nature. This book was released on 2023-02-11 with total page 396 pages. Available in PDF, EPUB and Kindle. Book excerpt: This second edition provides new and updated protocols that can be used for generation of knockout animals. Chapters guide the reader through basic protocols for three genome editing technologies, target design tools, and specific protocols for each animal. Written in the successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible protocols, and notes on troubleshooting and avoiding known pitfalls. Authoritative and cutting-edge, Genome Editing in Animals: Methods and Protocols, Second Edition aims to be a useful practical guide to researches to help further their study in this field.
Download or read book Protein Targeting Protocols written by Roger A. Clegg and published by Springer Science & Business Media. This book was released on 2008-02-04 with total page 331 pages. Available in PDF, EPUB and Kindle. Book excerpt: It is by no means a revelation that proteins are not uniformly distributed throughout the cell. As a result, the idea that protein molecules, because of the specificity with which they can engage in interactions with other proteins, may be aimed—via these interactions—at a restricted target, is a fundamental one in contemporary molecular life sciences. The target may be variously c- ceived as a specific molecule, a group of molecules, a structure, or a more generic type of intracellular environment. Because the concept of protein targeting is intuitive rather than expl- itly defined, it has been variously used by different groups of researchers in cell biology, biochemistry, and molecular biology. For those working in the field of intracellular signaling, an influential introduction to the topic was the seminal article by Hubbard & Cohen (TIBS [1993] 18, 172–177), which was based on the work of Cohen’s laboratory on protein phosphatases. Sub- quently, the ideas that they discussed have been further developed and extended by many workers to other key intermediaries in intracellular sign- ing, including protein kinases and a great variety of modulator and adaptor proteins.
