title: Optimization of methods for the genetic modification of human T cells.
abstract: CD4(+) T cells are not only critical in the fight against parasitic, bacterial and viral infections, but are also involved in many autoimmune and pathological disorders. Studies of protein function in human T cells are confined to techniques such as RNA interference (RNAi) owing to ethical reasons and relative simplicity of these methods. However, introduction of RNAi or genes into primary human T cells is often hampered by toxic effects from transfection or transduction methods that yield cell numbers inadequate for downstream assays. Additionally, the efficiency of recombinant DNA expression is frequently low because of multiple factors including efficacy of the method and strength of the targeting RNAs. Here, we describe detailed protocols that will aid in the study of primary human CD4(+) T cells. First, we describe a method for development of effective microRNA/shRNAs using available online algorithms. Second, we illustrate an optimized protocol for high efficacy retroviral or lentiviral transduction of human T-cell lines. Importantly, we demonstrate that activated primary human CD4(+) T cells can be transduced efficiently with lentiviruses, with a highly activated population of T cells receiving the largest number of copies of integrated DNA. We also illustrate a method for efficient lentiviral transduction of hard-to-transduce un-activated primary human CD4(+) T cells. These protocols will significantly assist in understanding the activation and function of human T cells and will ultimately aid in the development or improvement of current drugs that target human CD4(+) T cells.
authors.affiliation: Interdisciplinary Graduate Program in Immunology, University of Iowa, Iowa City, IA, USA.
authors.forename: Mahmood Y
authors.initials: MY
authors.lastname: Bilal
authors.affiliation: Interdisciplinary Graduate Program in Immunology, University of Iowa, Iowa City, IA, USA.
authors.forename: Aldo
authors.initials: A
authors.lastname: Vacaflores
authors.affiliation: Interdisciplinary Graduate Program in Immunology, University of Iowa, Iowa City, IA, USA.|Department of Microbiology, Carver College of Medicine, University of Iowa, Iowa City, IA, USA.
authors.forename: Jon Cd
authors.initials: JC
authors.lastname: Houtman
mesh_terms: D000465:Algorithms; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology*; D002478:Cells, Cultured; D019295:Computational Biology; D015316:Genetic Therapy; D005822:Genetic Vectors; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D008213:Lymphocyte Activation; D035683:MicroRNAs / Q000235:genetics*; D034622:RNA Interference; D012190:Retroviridae / Q000235:genetics*; D014161:Transduction, Genetic
chemical_list: D035683:MicroRNAs