An RNAi-based system for loss-of-function analysis identifies Raf1 as a crucial mediator of BCR-ABL-driven leukemogenesis
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An RNAi-based system for loss-of-function analysis identifies Raf1 as a crucial mediator of BCR-ABL-driven leukemogenesis. / Albers, Corinna; Illert, Anna L; Miething, Cornelius; Leischner, Hannes; Thiede, Melanie; Peschel, Christian; Duyster, Justus.
in: BLOOD, Jahrgang 118, Nr. 8, 25.08.2011, S. 2200-10.Publikationen: SCORING: Beitrag in Fachzeitschrift/Zeitung › SCORING: Zeitschriftenaufsatz › Forschung › Begutachtung
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TY - JOUR
T1 - An RNAi-based system for loss-of-function analysis identifies Raf1 as a crucial mediator of BCR-ABL-driven leukemogenesis
AU - Albers, Corinna
AU - Illert, Anna L
AU - Miething, Cornelius
AU - Leischner, Hannes
AU - Thiede, Melanie
AU - Peschel, Christian
AU - Duyster, Justus
PY - 2011/8/25
Y1 - 2011/8/25
N2 - 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.
AB - 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.
KW - Animals
KW - Base Sequence
KW - Cell Transformation, Neoplastic
KW - DNA Primers
KW - Female
KW - Gene Expression
KW - Gene Knockdown Techniques
KW - Genes, abl
KW - Genetic Vectors
KW - Leukemia, Myelogenous, Chronic, BCR-ABL Positive
KW - MAP Kinase Signaling System
KW - Mice
KW - Mice, Inbred BALB C
KW - Proto-Oncogene Proteins B-raf
KW - Proto-Oncogene Proteins c-raf
KW - RNA Interference
KW - Journal Article
KW - Research Support, Non-U.S. Gov't
KW - Validation Studies
U2 - 10.1182/blood-2010-10-309583
DO - 10.1182/blood-2010-10-309583
M3 - SCORING: Journal article
C2 - 21715303
VL - 118
SP - 2200
EP - 2210
JO - BLOOD
JF - BLOOD
SN - 0006-4971
IS - 8
ER -
