transfection of hek293t cells using non&#45;liposomal lipid transfection reagent for lentiviral production

Lentiviral Vector Preparation for In Vivo Gene Modulation 

Tissue-resident macrophages (M&#966;) are a heterogeneous population of phagocytic immune cells that sense and respond to invading pathogens1,2. In addition, they play an essential role in tissue development, remodeling, and maintaining homeostasis1,3. Many tissue M&#966; derive from yolk sac progenitors during embryogenesis and persist in the tissue throughout the life4,5. The phenotype and functions of these cells are dictated by collaborative and hierarchical interactions of specific transcription factors and the local microenvironment6,7,8,9. A growing understanding of this dependency increases the need for effective in vivo methods for gene manipulation of M&#966; within their physiologically relevant niche.
Lentiviral vectors are a frequently employed tool for the manipulation of nucleic acids in specific cell populations in vivo10,11,12, particularly due to their ability to infect both dividing and non-dividing cells and to stably integrate into host genome13,14. Over the last two decades, lentivirus delivery technology has been optimized, and alternative envelopes and synthetic promoters have been investigated to increase lineage-specific targeting8,15. Owing to its broad cell tropism, vesicular stomatitis virus envelop glycoprotein (VSV-G)16,17 has become the &#34;gold-standard&#34; envelope used in lentivirus technology.
In this protocol18, VSV-G pseudotyped lentiviral particles are employed to demonstrate targeted and effective delivery of short hairpin RNA (shRNA) and microRNA (miR) to mouse peritoneal M&#966; (pM&#966;) in vivo, at steady state19. Transgene expression was driven by the spleen focus forming virus (SFFV) promoter. Productive infection of cells was defined by expression of lentivirus-derived enhanced green fluorescent protein (GFP). Utilization of this approach allowed easy readout for in vivo lentivirus experiments to define the optimal dose and the experimental timeframe. Finally, in vivo lentiviral challenge of mice during thioglycolate-induced inflammation revealed the natural propensity for selective pM&#966; infection.

Author Spotlight: Exploring Macrophage Immunometabolism Through Lentiviral Vector&#45;Mediated Gene Manipulation

Lentiviral Vector Preparation for Efficient Gene and MicroRNA Modulation of Peritoneal Cavity Tissue&#45;Resident Macrophages In Vivo in Mice

Compared to other viral approaches for in vivo gene manipulation in macrophages, the use of lentiviral vectors offers a stable integration of the transgenic tissue macrophages, efficient transgene expression, and the largest vector size limit. When employing in vivo lentiviral manipulation of metabolic enzymes, we discovered notable functional changes in tissue resident macrophages. Therefore, we are currently investigating immuno metabolism and its role in preserving tissue specific functions, including the epigenetics and immune function.In the future, we would like to focus on defining how tissue macrophages affect other local cell populations during homeostasis and in disease models, and how these interactions can be employed for disease prevention and resolution.

Transfection of HEK293T Cells Using Non&#45;Liposomal Lipid Transfection Reagent for Lentiviral Production

To begin, add the lentiviral components in a 15-milliliter tube, and make the volume to 600 microliters with EC buffer. Add 36 microliters of enhancer solution, and using a one-milliliter pipette, mix repeatedly, drawing up and releasing a portion of the liquid back into the solution. After five minutes, add 120 microliters of transfection reagent and mix approximately 20 times, as demonstrated previously.Leave the tube at room temperature for 10 minutes. Then add 5.2 milliliters of cDMEM to the tube. Using a disposable transfer pipette, dropwise add the transfection mixture onto the HEK293T cells monolayer cultured in a T175 flask.Distribute the plasmid evenly through a gentle side-to-side rocking motion of the flask. Incubate the culture at 37 degrees Celsius, and 5%carbon dioxide for 48 hours. After incubation under a fluorescent microscope, confirm the effective transfection of HEK293T cells by observing GFP fluorescence.Now tilt the flask, and using a 25-milliliter serological stripette, collect the medium without disturbing the cell monolayer. Transfer the medium to a pre-labeled 50-milliliter tube and close the lid. Add 25 milliliters of warm cDMEM along the walls of the flask without disturbing the monolayer, and return the flask to the incubator.After 24 hours, collect the medium, add the decontamination solution into the flask, and place it horizontally for the next 24 hours.

Research

JoVE Journal

Immunology and Infection

Peritoneal tissue-resident macrophages have broad functions in the maintenance of homeostasis and are involved in pathologies within local and neighboring ...