Inducible microRNA expression by an all-in-one episomal vector system.
Here we describe an episomal, one-vector system which allows the generation of cell populations displaying homogenous, inducible gene inactivation by RNA interference in a one step procedure. A dual tet-repressor/activator system tightly controls a bi-directional promoter, which simultaneously drives expression of microRNAs and a fluorescent marker protein. We demonstrate the effectiveness of this vector by knockdown of p53 expression in a human cell line which resulted in the expected loss of G1-arrest after DNA damage. The generation of a cell pool homogenously expressing the ectopic microRNAs was achieved in 1 week without the need for viral infections. Induction of microRNA expression did not elicit an interferon response. Furthermore, the vector was adapted for convenient ligation-free transfer of microRNA cassettes from public libraries. This conditional knockdown-system should prove useful for many research and gene therapeutic applications.
Epanchintsev
Alexey
A
Molecular Oncology, Independent Max-Planck-Research Group, Max-Planck-Institute of Biochemistry, Am Klopferspitz 18, D-82152 Martinsried/Munich, Germany.
Jung
Peter
P
Menssen
Antje
A
Hermeking
Heiko
H
D045744:Cell Line, Tumor; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D006801:Humans; D035683:MicroRNAs / Q000096:biosynthesis / Q000235:genetics*; D010957:Plasmids / Q000235:genetics*; D034622:RNA Interference; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000096:biosynthesis / Q000235:genetics
D035683:MicroRNAs; D012097:Repressor Proteins; D015534:Trans-Activators; D016159:Tumor Suppressor Protein p53; C034811:tetracycline resistance-encoding transposon repressor protein