title: Stable Delivery of CCR5-Directed shRNA into Human Primary Peripheral Blood Mononuclear Cells and Hematopoietic Stem/Progenitor Cells via a Lentiviral Vector.
abstract: RNAi is a powerful tool to achieve suppression of a specific gene expression and therefore it has tremendous potential for gene therapy applications. A number of vector systems have been developed to express short-hairpin RNAs (shRNAs) to produce siRNAs within mammalian T-cells, primary hematopoietic stem/progenitor cells (HSPC), human peripheral blood mononuclear cells, and in animal model systems. Among these, vectors based on lentivirus backbones have significantly transformed our ability to transfer shRNAs into nondividing cells, such as HSPC, resulting in high transduction efficiencies. However, delivery and long-term expression of shRNAs should be carefully optimized for efficient knock down of target gene without causing cytotoxicity in mammalian cells. Here, we describe our protocols for the development of shRNA against a major HIV co-receptor/chemokine receptor CCR5 and the use of lentiviral vectors for stable shRNA delivery and expression in primary human PBMC and HSPC.
authors.affiliation: School of Nursing, University of California, Los Angeles, 188 BSRB, 615 Charles E Young Dr South, Los Angeles, CA, 90095, USA.|UCLA AIDS Institute, Los Angeles, CA, USA.
authors.forename: Saki
authors.initials: S
authors.lastname: Shimizu
authors.affiliation: School of Nursing, University of California, Los Angeles, 188 BSRB, 615 Charles E Young Dr South, Los Angeles, CA, 90095, USA.|UCLA AIDS Institute, Los Angeles, CA, USA.
authors.forename: Swati Seth
authors.initials: SS
authors.lastname: Yadav
authors.affiliation: School of Nursing, University of California, Los Angeles, 188 BSRB, 615 Charles E Young Dr South, Los Angeles, CA, 90095, USA. an@ucla.edu.|UCLA AIDS Institute, Los Angeles, CA, USA. an@ucla.edu.|Hematology-Oncology, The Department of Medicine, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA. an@ucla.edu.
authors.forename: Dong Sung
authors.initials: DS
authors.lastname: An
mesh_terms: D018952:Antigens, CD34 / Q000378:metabolism; D016503:Drug Delivery Systems / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics*; D057809:HEK293 Cells; D006412:Hematopoietic Stem Cells / Q000378:metabolism*; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D007963:Leukocytes, Mononuclear / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism*; D060888:Real-Time Polymerase Chain Reaction; D019713:Receptors, CCR5 / Q000378:metabolism*; D014161:Transduction, Genetic
chemical_list: D018952:Antigens, CD34; D034741:RNA, Small Interfering; D019713:Receptors, CCR5
keywords: 293-T cells; CCR5; CD34+ cells; Lentiviral vectors; PBMC; Transduction; Transfection; shRNA