High efficiency protein transduction of quiescent and proliferating primary hematopoietic cells.
J Biochem Biophys Methods
; 55(3): 251-8, 2003 Mar 28.
Article
em En
| MEDLINE
| ID: mdl-12706909
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.
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Base de dados:
MEDLINE
Assunto principal:
Transdução Genética
/
Proteínas Recombinantes de Fusão
/
Células-Tronco Hematopoéticas
/
Engenharia de Proteínas
/
Produtos do Gene tat
/
Regulação da Expressão Gênica
Tipo de estudo:
Evaluation_studies
Limite:
Humans
Idioma:
En
Revista:
J Biochem Biophys Methods
Ano de publicação:
2003
Tipo de documento:
Article
País de afiliação:
Reino Unido