Basic principles Basic principles involved in correcting a genetic defect are: Gene
replacement, i.e. replacement of a mutant gene with a normal gene. Gene
correction, i.e. correction of the mutated area (specific bases) of DNA leaving the
Author: Indu Khurana
Publisher: Elsevier India
Encouraged by the response to the first edition, this edition highlights the essential and relevant content of physiology with complete and balanced exposition of text with absolute clarity. With the balanced amalgamation of pure and applied text, authors aspire it to be an indispensable text for undergraduates and an authentic reference source for candidates preparing for PG entrance. Complete and up-to-date text with recent advances incorporated Illustrated by more than 1000 clear line diagrams Complemented with numerous tables and flowcharts for quick comprehension Balanced amalgamation of pure and applied text Highlights applied aspects of physiology in separate boxes Systematic organization of text to facilitate easy review Additional important information has been highlighted in the form of Important Notes Core competencies prescribed by the MCI are covered and competency codes are included in the text
In this review, we detail how DNA transposons could be emphasized to create a new tool for gene correction.
Author: Colette Bastie
Transposable elements (TEs) are mobile genetic sequences that are able to move in the genome from one location to another. TEs were first regarded as junk or selfish DNA, as they comprise the largest molecular class within most metazoan genomes having no genomic function. It was necessary to wait until whole genome sequencing to provide new insights about the origin, diversity, and impact of TEs on the genome function. Thus, due to advances in molecular technology, TEs have been shown to create new regulatory sequence networks. Although nowadays most TEs present in the human genome are silenced, particularly DNA transposons, it does not mean that these sequences are dead. In this review, we detail how DNA transposons could be emphasized to create a new tool for gene correction. DNA-based transposon vectors are derived from three models: Sleeping Beauty, piggyBac, and Tol2, which all work via a "cut-and-paste" mechanism where transposase enzyme is alone able to catalyze the transposition process, which means integrating the genes of interest in chromosomal DNA. Limitations and improvements of the systems are discussed, particularly the latest way to target a specific integration site, showing that the DNA transposon-derived system and its engineering, are powerful tools for gene correction.
Gruenert DC : Gene correction with small DNA fragments . Curr Res Molec Ther 1
: 607–613 ( 1998 ) . Gruenert DC : Opportunities and challenges in targeting
genes for therapy . Gene Ther 6 : 1347-1348 ( 1999 ) . Gruenert DC , Xu Z ,
Author: Silvia Garagna
Publisher: S Karger Ag
The house mouse holds a unique place among model organisms, representing an indispensable tool for many investigators, particularly in biomedical research. Mouse genetics encompasses nearly 100 years of research and culminated in the publication of the mouse genome sequence in 2002, from which the idea for this special issue arose. The issue features 37 peer-reviewed papers providing a wide-ranging overview of all major aspects of mouse genetics, from nuclear transfer and embryonic development to cancer, from reproduction to speciation and functional genomics technologies. Its aim is to present a suitable tool of investigation for the large population of researchers that use the mouse as a model animal. Covering a variety of topics, this publication will be a useful guide for scientists working in the fields of reproduction, development, evolution, cancer, toxicology, and cell biology as well as for beginners entering the world of mouse genetics.
PurposeWiskott-Aldrich syndrome (WAS) is an X linked disease caused by a defect in the WAS gene. Affected boys suffer from recurrent infections, eczema, bleeding, auto-immune symptoms and an increased risk of leukemia.
PurposeWiskott-Aldrich syndrome (WAS) is an X linked disease caused by a defect in the WAS gene. Affected boys suffer from recurrent infections, eczema, bleeding, auto-immune symptoms and an increased risk of leukemia. Since the WAS protein is involved in the coupling of the T cell receptor to the cytoskeleton, the absence of this protein results in a suboptimal T cell response to pathogens. In addition, we have shown that thymic T cell differentiation is affected which may explain both immune deficiency and autoimmunity (Laskowski et al). Current treatment consists of stem cell transplantation from an HLA matched donor. However, this frequently causes severe side-effects, especially acute and chronic graft-versus-host disease. We are currently developing a gene editing technique using WAS-targeted gRNA-CRISPR/Cas9 complexes and various donor constructs encoding the wild type WAS gene packaged in adeno associated virus 6 (AAV6) particles. We will validate this approach by evaluating the normalization of T cell repertoire development and antigen specific T cell responses.Materials and MethodsAn donor construct consisting of a left and right homology arm flanking a constitutive promoter pgk followed by a GFP encoding sequence was generated and transfected in 293T cells for AAV6 packaging. This approach is used to optimize the genetic correction protocol as shown in the figure. Cutting efficiency is evaluated on male donor cord blood using both Sanger sequencing with Tide analysis and NGS. Integration is measured by flowcytometry.ResultsWe optimized the electroporation protocol: electroporator, electroporation conditions, the amount of crispr/CAS9 guideRNA complex as well as the production of AAV6 particles. At this moment, we consistently obtain cutting efficiencies of 80% and efficiencies of homologous recombination of 45%. The latest results will be presented.ConclusionWe have shown that it is possible to efficiently target the WAS locus in hematopoietic precursor cells and to genetically correct about half of these cells.
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 ...
Author: Francesca Storici
Publisher: Humana Press
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.