title: An RNAi-based system for loss-of-function analysis identifies Raf1 as a crucial mediator of BCR-ABL-driven leukemogenesis.
abstract: Genetic loss-of-function studies in murine tumor models have been essential in the analysis of downstream mediators of oncogenic transformation. Unfortunately, these studies are frequently limited by the availability of genetically modified mouse strains. Here we describe a versatile method allowing the efficient expression of an oncogene and simultaneous knockdown of targets of interest (TOI) from a single retroviral vector. Both oncogene and TOI-specific miR30-based shRNA are under the control of the strong viral long terminal repeat promoter, resulting in a single shared RNA transcript. Using this vector in a murine syngeneic BM transplantation model for BCR-ABL-induced chronic myeloid leukemia, we find that oncogene expression and target knockdown in primary hematopoietic cells with this vector is efficient both in vitro and in vivo, and demonstrate that Raf1, but not BRAF, modulates BCR-ABL-dependent ERK activation and transformation of hematopoietic cells. This expression system could facilitate genetic loss-of-function studies and allow the rapid validation of potential drug targets in a broad range of oncogene-driven murine tumor models.
authors.affiliation: Department of Internal Medicine III, Klinikum Rechts der Isar, Technical University of Munich, Munich, Germany.
authors.forename: Corinna
authors.initials: C
authors.lastname: Albers
authors.forename: Anna L
authors.initials: AL
authors.lastname: Illert
authors.forename: Cornelius
authors.initials: C
authors.lastname: Miething
authors.forename: Hannes
authors.initials: H
authors.lastname: Leischner
authors.forename: Melanie
authors.initials: M
authors.lastname: Thiede
authors.forename: Christian
authors.initials: C
authors.lastname: Peschel
authors.forename: Justus
authors.initials: J
authors.lastname: Duyster
mesh_terms: D000818:Animals; D001483:Base Sequence; D002471:Cell Transformation, Neoplastic / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D005260:Female; D015870:Gene Expression; D055785:Gene Knockdown Techniques; D016313:Genes, abl; D005822:Genetic Vectors; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000201:enzymology / Q000209:etiology* / Q000235:genetics; D020935:MAP Kinase Signaling System; D051379:Mice; D008807:Mice, Inbred BALB C; D048493:Proto-Oncogene Proteins B-raf / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology; D019908:Proto-Oncogene Proteins c-raf / Q000037:antagonists & inhibitors* / Q000235:genetics* / Q000502:physiology; D034622:RNA Interference
chemical_list: D017931:DNA Primers; C482120:Braf protein, mouse; D048493:Proto-Oncogene Proteins B-raf; D019908:Proto-Oncogene Proteins c-raf