title: A tightly controlled conditional knockdown system using the Tol2 transposon-mediated technique.
abstract: BACKGROUND
  Gene knockdown analyses using the in utero electroporation method have helped reveal functional aspects of genes of interest in cortical development. However, the application of this method to analyses in later stages of brain development or in the adult brain is still difficult because the amount of injected plasmids in a cell decreases along with development due to dilution by cell proliferation and the degradation of the plasmids. Furthermore, it is difficult to exclude the influence of earlier knockdown effects.
  
  
  RESULTS
  We developed a tightly controlled conditional knockdown system using a newly constructed vector, pT2K-TBI-shRNAmir, based on a Tol2 transposon-mediated gene transfer methodology with the tetracycline-inducible gene expression technique, which allows us to maintain a transgene for a long period of time and induce the knockdown of the gene of interest. We showed that expression of the endogenous amyloid precursor protein (APP) was sharply decreased by our inducible, stably integrated knockdown system in PC12 cells. Moreover, we induced an acute insufficiency of Dab1 with our system and observed that radial migration was impaired in the developing cerebral cortex. Such inhibitory effects on radial migration were not observed without induction, indicating that our system tightly controlled the knockdown, without any expression leakage in vivo.
  
  
  CONCLUSIONS
  Our system enables us to investigate the brain at any of the later stages of development or in the adult by utilizing a knockdown technique with the aid of the in utero electroporation gene transfer methodology. Furthermore, we can perform knockdown analyses free from the influence of undesired earlier knockdown effects.
authors.affiliation: Division of Cell Biology and Neuroscience, Department of Morphological and Physiological Sciences, Faculty of Medical Sciences, University of Fukui, Fukui, Japan.
authors.forename: Tokuichi
authors.initials: T
authors.lastname: Iguchi
authors.forename: Hideshi
authors.initials: H
authors.lastname: Yagi
authors.forename: Chen-Chi
authors.initials: CC
authors.lastname: Wang
authors.forename: Makoto
authors.initials: M
authors.lastname: Sato
mesh_terms: D016564:Amyloid beta-Protein Precursor / Q000235:genetics; D000818:Animals; D001921:Brain / Q000254:growth & development*; D017931:DNA Primers / Q000235:genetics; D004251:DNA Transposable Elements / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D018274:Electroporation / Q000379:methods*; D005434:Flow Cytometry; D055785:Gene Knockdown Techniques / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000378:metabolism; D015151:Immunoblotting; D051379:Mice; D008813:Mice, Inbred ICR; D008856:Microscopy, Fluorescence; D034622:RNA Interference; D060888:Real-Time Polymerase Chain Reaction
chemical_list: D016564:Amyloid beta-Protein Precursor; C078201:Aplp1 protein, mouse; D017931:DNA Primers; D004251:DNA Transposable Elements; D018076:DNA, Complementary; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins