A novel method for long term bone marrow culture and genetic modification of murine neutrophils via retroviral transduction.
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.
Zemans
Rachel L
RL
Department of Medicine, National Jewish Health, Denver, CO, United States.
Briones
Natalie
N
Young
Scott K
SK
Malcolm
Kenneth C
KC
Refaeli
Yosef
Y
Downey
Gregory P
GP
Worthen
G Scott
GS
D000818:Animals; D017209:Apoptosis; D001853:Bone Marrow / Q000378:metabolism*; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation; D015870:Gene Expression; D006098:Granulocytes / Q000166:cytology / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D009504:Neutrophils / Q000166:cytology / Q000378:metabolism*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics*; D013997:Time Factors; D019076:Transgenes / Q000235:genetics*
D019253:Proto-Oncogene Proteins c-bcl-2