Stable GFP transduction of hard-to-transfect mouse macrophage RAW264.7 cells by a second-generation lentiviral vector

Salati, Monir; Abachi, Mansour; Sadeghizadeh, Majid; Soleimanpour-lichaei, Hamid Reza

Stable GFP transduction of hard-to-transfect mouse macrophage RAW264.7 cells by a second-generation lentiviral vector

Authors

Monir Salati ¹

Mansour Abachi ²

Majid Sadeghizadeh ¹

Hamid Reza Soleimanpour-lichaei ²

¹ Tarbiat Modares University

² National Institute of Genetic Engineering and Biotechnology (NIGEB)

Abstract

Introduction: Macrophages are considered a particularly challenging cell type to transfect. Given their importance as therapeutic targets, developing a successful transfection method for these cells is highly desirable.

Materials and Methods: The efficiency of lentiviral transfection was compared to three commercially transfection reagents (Xfect™ Transfection Reagent, FuGENE® HD, and Lipofectamine ^TM 3000) in RAW264.7 macrophage cells. Following optimization and production of lentiviral particles in 293T cells, RAW264.7 cells were infected with varying MOI. Transduction efficiency, cell viability, and metabolic activity were measured and compared to the transfection efficiency of the chemical methods.

Results: None of the three chemical transfection reagents successfully transfected RAW264.7 cells. In contrast, the lentiviral method achieved transduction even at the lowest concentration of viral stock, with a green fluorescent signal observable under a fluorescence microscope. Increasing the viral stock concentration and using higher MOIs (up to 30) significantly (p≤0.0001) increased transduction efficiency.

Discussion: Despite requiring more time and effort than chemical methods, lentiviral transduction exhibited superior efficiency in transfecting hard-to-transfect cells and further improvements were achieved through some modifications such as virus concentration, the use of polybrene, no viral freezing and O/N incubation with concentrated viral particles. Since other parameters, especially the use of retronectin and spinoculation, are effective on the efficiency of the virus infection process, it is suggested that they be considered in future studies and given the encouraging data of this research, this completed method can also be applied to other difficult-to-transfect cells, such as different types of stem cells or primary cells.

Keywords

Transfection

Transduction

RAW264.7

Difficult-to-transfect cells

Lentiviral vector

MOI

Subjects

Molecular biotechnology

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