Targeted gene disruption with engineered zinc-finger nucleases (ZFNs)

Methods Mol Biol. 2012;917:129-41. doi: 10.1007/978-1-61779-992-1_7.

Abstract

Reverse genetics in Xenopus has been limited to knockdown strategies using antisense morpholino oligonucleotides (MOs). Recently, engineered zinc-finger nucleases have been used to induce targeted mutations resulting in null alleles. Zinc-finger nuclease (ZFN) technology has been adapted to induce null mutations in many systems previously refractory to targeted gene inactivation. Here we provide a general protocol for inducing targeted mutations in Xenopus tropicalis using ZFNs, a method to detect resulting mutations, and the steps to generate homozygous mutant embryos.

MeSH terms

  • Animals
  • Animals, Inbred Strains
  • Chorionic Gonadotropin / administration & dosage
  • Cloning, Molecular
  • DNA Mutational Analysis
  • Deoxyribonucleases / chemistry
  • Deoxyribonucleases / genetics*
  • Embryo, Nonmammalian / physiology
  • Female
  • Fertilization
  • Germ-Line Mutation
  • Homozygote
  • INDEL Mutation
  • Male
  • Mutagenesis
  • Protein Engineering
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / genetics
  • Reproductive Control Agents / administration & dosage
  • Reverse Genetics*
  • Xenopus / genetics*
  • Zinc Fingers

Substances

  • Chorionic Gonadotropin
  • Recombinant Fusion Proteins
  • Reproductive Control Agents
  • Deoxyribonucleases