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Synthetic neomycin‐kanamycin phosphotransferase, type II coding sequence for gene targeting in mammalian cells
Author(s) -
Jin SeungGi,
Mann Jeffrey R.
Publication year - 2005
Publication title -
genesis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.093
H-Index - 110
eISSN - 1526-968X
pISSN - 1526-954X
DOI - 10.1002/gene.20131
Subject(s) - biology , gene , coding region , neomycin , genetics , cpg site , microbiology and biotechnology , gene expression , dna methylation , antibiotics
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. genesis 42:207–209, 2005. © 2005 Wiley‐Liss, Inc.