Red/ET recombination with chimeric oligonucleotides allows rapid generation of BAC transgenes harboring full-length or truncated huntingtin cDNA

Biotechniques. 2012 Jul 1;53(1). doi: 10.2144/000113908.

Abstract

Huntington's disease (HD) is a fatal neurodegenerative disorder that is caused by a CAG repeat expansion encoding a polyglutamine tract in the huntingtin (htt) gene. None of the existing HD mouse models recapitulate the exact disease symptoms and course as it is seen in humans and the generation of further HD disease models is challenging because of the size and complexity of the htt gene locus. Starting from a single substrate plasmid harboring human htt cDNA comprising 98 glutamine (Q) residues, we applied Red/ET recombination to generate four BDNF-BAC transgenes harboring full-length or truncated (N171) htt cDNA comprising 98 or 15 Q residues. BDNF (brain-derived neurotrophic factor) is expressed in the cortical neurons projecting to the striatal medium spiny neurons, and was used to direct htt transgene expression to investigate the contribution of these cell types to HD.

Keywords: BAC transgene; BDNF; Huntington's disease; Red/ET recombination; counterselection; huntingtin; recombineering.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Chromosomes, Artificial, Bacterial / genetics*
  • DNA, Complementary / genetics
  • Genetic Engineering / methods*
  • Humans
  • Huntingtin Protein
  • Huntington Disease
  • Mice
  • Nerve Tissue Proteins / genetics*
  • Nerve Tissue Proteins / metabolism
  • Oligonucleotides
  • Recombination, Genetic / genetics*

Substances

  • DNA, Complementary
  • HTT protein, human
  • Huntingtin Protein
  • Nerve Tissue Proteins
  • Oligonucleotides