Generation of topically transgenic rats by in utero electroporation and in vivo bioluminescence screening

J Vis Exp. 2013 Sep 24;(79):e50146. doi: 10.3791/50146.

Abstract

In utero electroporation (IUE) is a technique which allows genetic modification of cells in the brain for investigating neuronal development. So far, the use of IUE for investigating behavior or neuropathology in the adult brain has been limited by insufficient methods for monitoring of IUE transfection success by non-invasive techniques in postnatal animals. For the present study, E16 rats were used for IUE. After intraventricular injection of the nucleic acids into the embryos, positioning of the tweezer electrodes was critical for targeting either the developing cortex or the hippocampus. Ventricular co-injection and electroporation of a luciferase gene allowed monitoring of the transfected cells postnatally after intraperitoneal luciferin injection in the anesthetized live P7 pup by in vivo bioluminescence, using an IVIS Spectrum device with 3D quantification software. Area definition by bioluminescence could clearly differentiate between cortical and hippocampal electroporations and detect a signal longitudinally over time up to 5 weeks after birth. This imaging technique allowed us to select pups with a sufficient number of transfected cells assumed necessary for triggering biological effects and, subsequently, to perform behavioral investigations at 3 month of age. As an example, this study demonstrates that IUE with the human full length DISC1 gene into the rat cortex led to amphetamine hypersensitivity. Co-transfected GFP could be detected in neurons by post mortem fluorescence microscopy in cryosections indicating gene expression present at ≥6 months after birth. We conclude that postnatal bioluminescence imaging allows evaluating the success of transient transfections with IUE in rats. Investigations on the influence of topical gene manipulations during neurodevelopment on the adult brain and its connectivity are greatly facilitated. For many scientific questions, this technique can supplement or even replace the use of transgenic rats and provide a novel technology for behavioral neuroscience.

Publication types

  • Research Support, Non-U.S. Gov't
  • Video-Audio Media

MeSH terms

  • Animals
  • Cerebral Cortex / embryology
  • Cerebral Cortex / physiology*
  • DNA / administration & dosage
  • DNA / genetics
  • Electroporation / methods*
  • Embryo, Mammalian
  • Female
  • Hippocampus / embryology
  • Hippocampus / physiology*
  • Injections, Intraventricular
  • Luciferases / biosynthesis
  • Luciferases / chemistry
  • Luciferases / genetics
  • Luminescent Measurements / methods*
  • Pregnancy
  • Rats
  • Rats, Transgenic
  • Transfection / methods*

Substances

  • DNA
  • Luciferases