Abstract
Over the past two decades, researchers have struggled to efficiently express foreign DNA in primary macrophages, impeding research progress. The applications of lipofection, electroporation, microinjection, and viral-mediated transfer typically result in disruptions in macrophage differentiation and function, low expression levels of exogenous proteins, limited efficiency and high cell mortality. In this report, after extensive optimization, we present a method of expressing large tagged proteins at high efficiency, consistency, and low cost using lentiviral infection. This method utilizes laboratory-propagated second generation plasmids to produce efficient virus that can be stored for later use. The expression of proteins up to 150 kDa in size is achieved in 30-70% of cells while maintaining normal macrophage differentiation and morphology as determined by fluorescence microscopy and Western blot analysis. This manuscript delineates the reagents and methods used to produce lentivirus to express exogenous DNA in murine bone marrow-derived macrophages sufficient for single cell microscopy as well as functional assays requiring large numbers of murine bone marrow-derived macrophages.
Highlights
Macrophages are white blood cells, which are essential components of the immune system
This study reported high expression efficiency in murine macrophage cell lines and bone marrow-derived macrophages, multiple rounds of cell sorting was required to achieve this, which can have a detrimental impact on cell viability [14]
We report here an efficient, consistent, inexpensive protocol of exogenous DNA expression in primary murine bone marrow-derived macrophages, using lentivirus transduction
Summary
Macrophages are white blood cells (leukocytes), which are essential components of the immune system. MRNA was observed to have GFP expression efficiencies up to 75% with little effect on cell viability, as evaluated by flow cytometry and Western blot analysis [8] Another group showed success in using the nucleofection technique to transfect shRNA and DNA constructs into a murine macrophage cell line in two different studies [9, 10]. We report here an efficient, consistent, inexpensive protocol of exogenous DNA expression in primary murine bone marrow-derived macrophages, using lentivirus transduction This second generation system would require two recombination events during viral production for release of replicating virus and is considered by the CDC to be a BSL-2 level activity, suitable for most tissue culture containment hoods. Rotor PR70) 99 Fluorescence Microscope: (Nikon Eclipse E800 fluorescence microscope equipped with a Hamamatsu ORCA-ER digital camera, Leica SP-5 Laser Scanning Confocal fluorescence microscope)
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