Lentiviral vector preparation for efficient gene and microRNA modulation of peritoneal cavity tissue-resident macrophages in vivo in mice

Gurney, Mark ORCID: https://orcid.org/0000-0003-1119-6638, Davies, Luke C., Jones, Ruth E., Bart, Valentina M., Jenkins, Robert H. ORCID: https://orcid.org/0000-0001-8500-9044, Brennan, Paul ORCID: https://orcid.org/0000-0001-8792-0499, Taylor, Philip R. ORCID: https://orcid.org/0000-0003-0163-1421 and Czubala, Magdalena A. ORCID: https://orcid.org/0000-0001-9881-1095 2024. Lentiviral vector preparation for efficient gene and microRNA modulation of peritoneal cavity tissue-resident macrophages in vivo in mice. Journal of Visualized Experiments 204 , e64926. 10.3791/64926

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Abstract

Peritoneal tissue-resident macrophages have broad functions in the maintenance of homeostasis and are involved in pathologies within local and neighboring tissues. Their functions are dictated by microenvironmental cues; thus, it is essential to investigate their behavior in an in vivo physiological niche. Currently, specific peritoneal macrophage-targeting methodologies employ whole-mouse transgenic models. Here, a protocol for effective in vivo modulation of mRNA and small RNA species (e.g., microRNA) expression in peritoneal macrophages using lentivirus particles is described. Lentivirus preparations were made in HEK293T cells and purified on a single sucrose layer. In vivo validation of lentivirus effectivity following intraperitoneal injection revealed predominant infection of macrophages restricted to local tissue. Targeting of peritoneal macrophages was successful during homeostasis and thioglycolate-induced peritonitis. The limitations of the protocol, including low-level inflammation induced by intraperitoneal delivery of lentivirus and time restrictions for potential experiments, are discussed. Overall, this study presents a quick and accessible protocol for the rapid assessment of gene function in peritoneal macrophages in vivo.

Item Type:	Article
Date Type:	Publication
Status:	Published
Schools:	Medicine
Publisher:	MyJove Corporation
ISSN:	1940-087X
Date of First Compliant Deposit:	28 March 2024
Date of Acceptance:	29 September 2023
Last Modified:	06 Aug 2024 18:06
URI:	https://orca.cardiff.ac.uk/id/eprint/167615

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