Lea Nicholas C, Buggins Andrea G S, Orr Stephen J, Mufti Ghulam J, Thomas N Shaun B
Department of Haematological Medicine, Guy's, King's and St Thomas' School of Medicine, Rayne Institute, 123 Coldharbour Lane, London, SE5 9NU, UK.
J Biochem Biophys Methods. 2003 Mar 28;55(3):251-8. doi: 10.1016/s0165-022x(03)00077-0.
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.
原代造血细胞对DNA转染方法相对不敏感。当它们处于静止或终末分化状态且不再能够分裂时尤其如此。然而，完整蛋白质可通过蛋白质转导引入此类细胞。我们对其他研究者使用的HIV-TAT蛋白的蛋白质转导结构域（PTD）进行了改造。以绿色荧光蛋白（GFP）作为报告基因，我们发现与天然HIV TAT结构域相比，这个新序列能使重组融合蛋白更有效地转导到多种受试造血细胞中。对于外周血CD34+细胞、树突状细胞、粒细胞、单核细胞和淋巴细胞均是如此，所有这些细胞均处于静止或终末分化状态。此外，我们能够转导急性髓细胞白血病（AML）患者的成髓细胞。在所有受试细胞类型中，转导效率几乎达到100%。如在静止T淋巴细胞上所测试的，在添加PTD或TAT-GFP融合蛋白后15 - 30秒转导达到最大值。这种方法将使我们能够研究多种基因产物在以前对基因转染研究有抗性的细胞类型中的作用。
