Extended core sequences from the cHS4 insulator are necessary for protecting retroviral vectors from silencing position effects

Hum Gene Ther. 2007 Apr;18(4):333-43. doi: 10.1089/hum.2007.021.


The prototypic chromatin insulator cHS4 has proven effective at reducing repressive chromosomal position effects on retroviral vector expression. We report here studies designed to identify the minimal chicken hypersensitive site-4 (cHS4) sequences necessary for this activity. Using a gammaretroviral reporter vector and expression analysis in cell lines and primary mouse hematopoietic progenitor colonies, we found that a 250-bp core fragment reported to contain most of the cHS4 insulating activity failed to prevent silencing when used alone, although some barrier activity was observed when this fragment was combined with a 790-bp, but not 596-bp, spacer. Similar studies showed that four copies of a 90-bp fragment containing the cHS4 enhancer-blocking activity actually repressed vector green fluorescent protein (GFP) expression. In contrast, a 400-bp fragment containing the 250-bp core plus 3' flanking sequences protected vector expression to the same degree as the full-length 1.2-kb fragment. The 400-bp fragment activity was confirmed in a lentiviral vector expressing human beta-globin in murine erythroid leukemia (MEL) cells. Taken together, these studies indicate that the insulating activity of the 250-bp cHS4 core can be influenced by distance, and identify an extended core element that confers full barrier activity in the setting of two different classes of retroviral vectors.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, N.I.H., Intramural

MeSH terms

  • Animals
  • Base Sequence
  • DNA / genetics
  • Gene Silencing*
  • Genetic Vectors / genetics*
  • Globins / genetics
  • Green Fluorescent Proteins / analysis
  • Green Fluorescent Proteins / antagonists & inhibitors
  • Green Fluorescent Proteins / genetics
  • Hematopoietic Stem Cells / metabolism
  • Insulator Elements / genetics*
  • Lentivirus / genetics*
  • Mice
  • NIH 3T3 Cells
  • Sequence Analysis, DNA
  • Transduction, Genetic


  • Green Fluorescent Proteins
  • Globins
  • DNA