Genetic Modification of Hematopoietic Stem Cells: Methods and Protocols(506 Methods in Molecular Biology)

Gene Transfer into Hematopoietic Cells: From Basic Science to Clinical Application Christopher Baum 1. The Potential of Gene Transfer into Hematopoietic Vectors with the potential for stable transgene integration are Cells widely used in basic hematology and clinical trials of gene me- cine. In basic research, both gain-of-function and loss-of-fu- tion situations of individual genes can be created by gene transfer, leading to a wide range of applications in developmental biology, stem cell biology, immunology, leukemia research, and human genetics. With the first evidence of successful modification of murine hematopoietic cells using retroviral gene vectors (1, 2), researchers have also explored the therapeutic potential of this approach. To date, the emerging discipline of gene therapy is a highly diversified field that offers entirely novel approaches to treat a great variety of human diseases (3). All hematopoietic cell types are of major interest in this context, since the modification of the hematopoietic stem cell population may potentially give rise to a completely transgenic hematopoiesis with the potential to cure genetic disorders or fight severe chronic infections, and the targeting of mature cells such as lymphocytes or antigen-p- senting dendritic cells offers all types of transient and semiper- nent modifications of the immune system. The unifying principle of gene medicine is the need to transfer complex nucleic acids cells that do not contribute to the germline (somatic cells).

Table of Contents:
Immunomagnetic Enrichment of Human and Mouse Hematopoietic Stem Cells for Gene Therapy Applications.- Isolation of Human and Mouse Hematopoietic Stem Cells.- Murine Hematopoietic Stem Cell Transduction Using Retroviral Vectors.- Genetic Modification of Human Hematopoietic Cells: Preclinical Optimization of Oncoretroviral-mediated Gene Transfer for Clinical Trials.- Short-Term Culture of Human CD34+ Cells for Lentiviral Gene Transfer.- T Cell Culture for Gammaretroviral Transfer.- Retroviral Transduction of Murine Primary T Lymphocytes.- Lentiviral Vector Gene Transfer into Human T Cells.- DNA Transposons for Modification of Human Primary T Lymphocytes.- Retroviral Gene Transfer into Primary Human Natural Killer Cells.- Lentiviral Vector-Mediated Genetic Programming of Mouse and Human Dendritic Cells.- In Situ (In Vivo) Gene Transfer into Murine Bone Marrow Stem Cells.- In Vivo and Ex Vivo Gene Transfer in Thymocytes and Thymocyte Precursors.- Design and Production of Retro- and Lentiviral Vectors for Gene Expression in Hematopoietic Cells.- Knock-Down of Gene Expression in Hematopoietic Cells.- The Use of Retroviral Vectors for tet-Regulated Gene Expression in Cell Populations.- Detection of Replication Competent Retrovirus and Lentivirus.- Release Testing of Retroviral Vectors and Gene-Modified Cells.- Copy Number Determination of Genetically-Modified Hematopoietic Stem Cells.- Tissue Procurement for Molecular Studies Using Laser-Assisted Microdissection.- Leukemia Diagnosis in Murine Bone Marrow Transplantation Models.- Humanized Mouse Models to Study the Human Haematopoietic Stem Cell Compartment.- Canine Models of Gene-Modified Hematopoiesis.- Detection of Retroviral Integration Sites by Linear Amplification-Mediated PCR and Tracking of Individual Integration Clonesin Different Samples.- Retroviral Insertion Site Analysis in Dominant Haematopoietic Clones.- Tracking Gene-Modified T Cells In Vivo.- DNA Microarray Studies of Hematopoietic Subpopulations.- Quantification of Genomic Mutations in Murine Hematopoietic Cells.- Proteomics Studies After Hematopoietic Stem Cell Transplantation.- Spectral Karyotyping and Fluorescence In Situ Hybridization of Murine Cells.- Database Setup for Preclinical Studies of Gene-Modified Hematopoiesis.- The US and EU Regulatory Perspectives on the Clinical Use of Hematopoietic Stem/Progenitor Cells Genetically Modified Ex Vivo by Retroviral Vectors.

Review :
From the reviews: "This book provides a unique and comprehensive resource of protocols for the genetic modification of various hematopoietic cell types and up-to-date procedures for molecular and systemic monitoring. ! This book is a great help for the design of excellent research in basic hematology, oncology, genetics, and immunology, and also promote the implementation of investigator-driven clinical studies using gene-modified hematopoietic cells. The book represents an outstanding work, which should not be missed in all biomedical research laboratories dealing with gene therapy of hematopoietic cells." (C. Altaner, Neoplasma, June, 2009) "This book provides a unique and comprehensive resource of protocols for the genetic modification of various hematopoietic cell types and up-to-date procedures for molecular and systemic monitoring. ! This book is a great help for the design of excellent research in basic hematology, oncology, genetics, and immunology, and also promote the implementation of investigator-driven clinical studies using gene-modified hematopoietic cells. The book represents an outstanding work, which should not be missed in all biomedical research laboratories dealing with gene therapy of hematopoietic cells." (C. Altaner, Neoplasma, April, 2009) "Haematopoietic stem cells are attractive targets for gene therapy. ! Each chapter provides a brief review of the area, followed by a step-by-step guide to undertaking experiments and includes details of reagents required and a list of useful notes or comments of issues that may arise. Comprehensive reference citations provide sources of further information when required. The book should provide a road map for researchers, clinical investigators and regulators involved in modifying haematopoietic cells." (Waseem Qasim, Human Genetics, Vol. 128, May, 2010)