Transduction of Murine Hematopoietic Stem Cells with Tetracycline-regulated Lentiviral Vectors.
Tetracycline-regulated integrating vectors allow pharmacologically controlled genetic modification of murine and human hematopoietic stem cells (HSCs). This approach combines the stable transgene insertion into a host genome with the opportunity for time- and dose-controlled reversible transgene expression in HSCs. Here, we describe the step-by-step protocol for transduction of murine stem-cell enriched populations of bone marrow cells, such as lineage negative cells (Lin(-)), with a lentiviral vector expressing the enhanced green fluorescent protein (EGFP) under the control of the tetracycline-regulated promoter. This chapter explains how to establish in vitro and in vivo systems to study transgene dose-dependent mechanisms affecting cell fate decisions of genetically modified hematopoietic cells.
Institute of Experimental Hematology, OE6960, Hannover Medical School, Carl-Neuberg-Straße 1, 30625, Hannover, Germany.
Maike
M
Stahlhut
Institute of Experimental Hematology, OE6960, Hannover Medical School, Carl-Neuberg-Straße 1, 30625, Hannover, Germany.|Division of Hematology/Oncology, Boston Children's Hospital, Harvard Medical School, Boston, MA, USA.
Axel
A
Schambach
Institute of Experimental Hematology, OE6960, Hannover Medical School, Carl-Neuberg-Straße 1, 30625, Hannover, Germany. Kustikova.Olga@mh-hannover.de.
Olga S
OS
Kustikova
D000818:Animals; D002454:Cell Differentiation / Q000235:genetics; D019070:Cell Lineage; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000187:drug effects; D006801:Humans; D016086:Lentivirus / Q000235:genetics; D051379:Mice; D011401:Promoter Regions, Genetic; D013752:Tetracycline / Q000494:pharmacology; D014161:Transduction, Genetic / Q000379:methods*
D013752:Tetracycline
Dose-dependent transgene expression; Gene transfer; Murine hematopoietic stem cells; Tetracycline-regulated lentiviral vectors; Transduction