Morpholino-mediated gene knockdown in mammalian organ culture

Methods Mol Biol. 2012:886:305-9. doi: 10.1007/978-1-61779-851-1_27.

Abstract

We examined the role of semaphorin3a in ureteric bud (UB) branching morphogenesis using mouse metanephric organ culture [Tufro et al. (Mech Dev 125:558-568, 2008)]. In vitro UB injection of Sema3a antisense morpholino resulted in increased branching morphogenesis. Cellular and tissue uptake of oligonucleotides was facilitated by a peptide-mediated method. Our findings were validated by in vitro translation and in Sema3a null mice. This chapter describes a method to perfuse the UB lumen with fluorescein-labeled oligonucleotides bound to a peptide carrier.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Drug Carriers / chemistry
  • Gene Knockdown Techniques / methods*
  • Kidney / embryology*
  • Kidney / metabolism
  • Mice
  • Microscopy, Confocal / methods
  • Molecular Sequence Data
  • Morpholinos / administration & dosage*
  • Morpholinos / genetics
  • Oligonucleotides, Antisense / administration & dosage*
  • Oligonucleotides, Antisense / genetics
  • Organ Culture Techniques / methods*
  • Peptides / chemistry
  • Perfusion / methods
  • Semaphorin-3A / genetics*

Substances

  • Drug Carriers
  • Morpholinos
  • Oligonucleotides, Antisense
  • Peptides
  • Semaphorin-3A