1. ¹Department of Chemical and Biomolecular Engineering, Institute for Medicine and Engineering, University of Pennsylvania, Philadelphia, PA, USA
2. ²FM Kirby Center for Molecular Ophthalmology, Scheie Eye Institute, 305 Stellar-Chance Laboratory, University of Pennsylvania, Philadelphia, PA, USA
3. ³Department of Medicine, Hematology/Oncology, Abramson Cancer Center, University of Pennsylvania, Philadelphia, PA, USA

Correspondence: Dr EA Pierce, FM Kirby Center for Molecular Ophthalmology, Scheie Eye Institute, 305 Stellar-Chance Laboratory, 422 Curie Blvd, University of Pennsylvania, Philadelphia, PA 19104, USA. E-mail: epierce@mail.med.upenn.edu

Received 15 January 2006; Revised 30 May 2006; Accepted 20 July 2006; Published online 5 October 2006.

Abstract

Single-stranded oligodeoxynucleotide (ssODN) gene targeting may facilitate animal model creation and gene repair therapy. Lipofection of ssODN can introduce point mutations into target genes. However, typical efficiencies in mouse embryonic stem cells (ESC) are <10^-4, leaving corrections too rare to effectively identify. We developed ESC lines with an integrated mutant neomycin resistance gene (Tyr22Ter). After targeting with ssODN, repaired cells survive selection in G418. Correction efficiencies varied with different lipofection procedures, clonal lines, and ssODN designs, ranging from 1 to 100 corrections per million cells plated. Uptake studies using cell sorting of Cy5-labelled ssODN showed 40% of the corrections concentrated in the best transfected 22% of cells. Four different basepair mismatches were tested and results show that the base-specificity of the mismatch is critical. Dual mismatch ssODN also showed mismatch preferences. These ESC lines may facilitate development of improved ssODN targeting technologies for either animal production or ex vivo gene therapy.