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A novel method for long term bone marrow culture and genetic modification of murine neutrophils via retroviral transduction.

Abstract：

:Neutrophils are a critical component of the innate immune response to invading microbial pathogens. However, an excessive and/or prolonged neutrophil response can result in tissue injury that is thought to underlie the pathogenesis of various inflammatory diseases. The development of novel therapeutic strategies for inflammatory diseases depends on an improved understanding of regulation of neutrophil function. However, investigations into neutrophil function have been constrained in part by the difficulty of genetically modifying neutrophils using current techniques. To overcome this, we have developed a novel method for the genetic modification of murine bone marrow derived progenitor cells using retroviral transduction followed by long term bone marrow culture to generate mature neutrophils. These neutrophils are functionally mature as determined by morphology, surface marker (Gr1, CD11b, CD62L and CXCR2) expression, and functional attributes including the ability to generate superoxide, exocytose granule contents, chemotax, and phagocytose and kill bacteria. Further, the in vitro matured neutrophils are capable of migrating to an inflammatory site in vivo. We utilized this system to express the Bcl-2 transgene in mature neutrophils using the retroviral vectors pMIG and pMIT. Bcl-2 overexpression conferred a substantial delay in spontaneous apoptosis of neutrophils as assessed by annexin V and 7-amino-actinomycin D (7AAD) staining. Moreover, Bcl-2 overexpression did not alter granulopoiesis, as assessed by morphology and surface marker expression. This system enables the genetic manipulation of progenitor cells that can be differentiated in vitro to mature neutrophils that are functional in vitro and in vivo.

journal_name

J Immunol Methods

journal_title

Journal of immunological methods

authors

Zemans RL,Briones N,Young SK,Malcolm KC,Refaeli Y,Downey GP,Worthen GS

doi

10.1016/j.jim.2008.10.004

subject

Has Abstract

pub_date

2009-01-30 00:00:00

pages

102-15

issue

eissn

0022-1759

issn

1872-7905

pii

S0022-1759(08)00316-5

journal_volume

340

pub_type

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A novel method for long term bone marrow culture and genetic modification of murine neutrophils via retroviral transduction.