title: High efficiency protein transduction of quiescent and proliferating primary hematopoietic cells.
abstract: Primary hematopoietic cells are relatively refractory to DNA transfection methodologies. This is particularly so when they are quiescent or terminally differentiated and no longer able to divide. However, whole proteins can be introduced into such cells by protein transduction. We have modified the protein transduction domain (PTD) from the HIV-TAT protein used by other investigators. Using green fluorescent protein (GFP) as a reporter, we show that this new sequence allows more efficient transduction of recombinant fusion protein into a variety of hematopoietic cells tested compared with the native HIV TAT domain. This is true for peripheral blood CD34+ cells, dendritic cells, granulocytes, monocytes and lymphocytes all of which are quiescent or terminally differentiated. Furthermore, we were able to transduce myeloblasts from patients with acute myeloid leukemia (AML). In all cell types tested transduction efficiency was almost 100%. Transduction is maximal 15-30 s after addition of PTD or TAT-GFP fusion proteins as tested on quiescent T lymphocytes. This method will allow us to study of the effects of a variety of gene products in cell types that were previously resistant to gene transfection studies.
authors.affiliation: Department of Haematological Medicine, Guy's, King's and St Thomas' School of Medicine, Rayne Institute, 123 Coldharbour Lane, London, SE5 9NU, UK. nicholas.lea@kcl.ac.uk
authors.forename: Nicholas C
authors.initials: NC
authors.lastname: Lea
authors.forename: Andrea G S
authors.initials: AG
authors.lastname: Buggins
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Orr
authors.forename: Ghulam J
authors.initials: GJ
authors.lastname: Mufti
authors.forename: N Shaun B
authors.initials: NS
authors.lastname: Thomas
mesh_terms: D002455:Cell Division; D005786:Gene Expression Regulation / Q000502:physiology*; D015696:Gene Products, tat / Q000235:genetics* / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D008164:Luminescent Proteins; D015202:Protein Engineering / Q000379:methods*; D011786:Quality Control; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics; D014161:Transduction, Genetic / Q000379:methods*
chemical_list: D015696:Gene Products, tat; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins