title: Lentiviral shRNA silencing of murine bone marrow cell CCR2 leads to persistent knockdown of CCR2 function in vivo.
abstract: A major barrier in hematopoietic gene function studies is posed by the laborious and time-consuming generation of knockout mice with an appropriate genetic background. Here we present a novel lentivirus-based strategy for the in situ generation of hematopoietic knockdowns. A short hairpin RNA (shRNA) was designed targeting murine CC-chemokine receptor 2 (CCR2), which was able to specifically blunt CCR2 expression at the mRNA, protein, and functional levels in vitro. Reconstitution of irradiated recipient mice with autologous bone marrow that had been ex vivo transduced with shRNA lentivirus led to persistent down-regulation of CCR2 expression, which translated into a 70% reduction in CCR2-dependent recruitment of macrophages to an inflamed peritoneal cavity without noticeable side effects on related chemokine receptors or general inflammation status. These findings clearly demonstrate the potential of shRNA lentivirus-infected bone marrow transplantation as a rapid and effective method to generate hematopoietic knockdowns for leukocyte gene function studies.
authors.affiliation: Division of Biopharmaceutics, Leiden/Amsterdam Center for Drug Research, Gorlaeus Laboratories, Leiden University, Einsteinweg 55, 2333 CC Leiden, The Netherlands. i.bot@lacdr.leidenuniv.nl
authors.forename: Ilze
authors.initials: I
authors.lastname: Bot
authors.forename: Jian
authors.initials: J
authors.lastname: Guo
authors.forename: Miranda
authors.initials: M
authors.lastname: Van Eck
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Van Santbrink
authors.forename: Pieter H E
authors.initials: PH
authors.lastname: Groot
authors.forename: Reeni B
authors.initials: RB
authors.lastname: Hildebrand
authors.forename: Jurgen
authors.initials: J
authors.lastname: Seppen
authors.forename: Theo J C
authors.initials: TJ
authors.lastname: Van Berkel
authors.forename: Erik A L
authors.initials: EA
authors.lastname: Biessen
mesh_terms: D000818:Animals; D001854:Bone Marrow Cells; D016026:Bone Marrow Transplantation; D002633:Chemotaxis; D015536:Down-Regulation; D016086:Lentivirus / Q000235:genetics*; D008264:Macrophages; D008722:Methods; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D023421:Models, Animal; D034622:RNA Interference; D012333:RNA, Messenger / Q000187:drug effects; D034741:RNA, Small Interfering / Q000138:chemical synthesis / Q000235:genetics* / Q000494:pharmacology; D054390:Receptors, CCR2; D019707:Receptors, Chemokine / Q000172:deficiency* / Q000187:drug effects / Q000235:genetics; D014161:Transduction, Genetic
chemical_list: C515335:Ccr2 protein, mouse; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D054390:Receptors, CCR2; D019707:Receptors, Chemokine