Synthetic neomycin-kanamycin phosphotransferase, type II coding sequence for gene targeting in mammalian cells

Seung Gi Jin, Jeffrey R. Mann

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1 Citation (Scopus)

Abstract

The bacterial neomycin-kanamycin phosphotransferase, type II enzyme is encoded by the neo gene and confers resistance to aminoglycoside drugs such as neomycin and kanamycin-bacterial selection and G418-eukaryotic cell selection. Although widely used in gene targeting in mouse embryonic stem cells, the neo coding sequence contains numerous cryptic splice sites and has a high CpG content. At least the former can cause unwanted effects in cis at the targeted locus. We describe a synthetic sequence, sneo, which encodes the same protein as that encoded by neo. This synthetic sequence has no predicted splice sites in either strand, low CpG content, and increased mammalian codon usage. In mouse embryonic stem cells sneo expressability is similar to neo. The use of sneo in gene targeting experiments should substantially reduce the probability of unwanted effects in cis due to splicing, and perhaps CpG methylation, within the coding sequence of the selectable marker.

Original languageEnglish
Pages (from-to)207-209
Number of pages3
Journalgenesis: The Journal of Genetics and Development
Volume42
Issue number3
DOIs
Publication statusPublished - 1 Jul 2005
Externally publishedYes

Keywords

  • Assembly PCR
  • DNA methylation
  • Gene targeting
  • Neomycin
  • Splicing

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