Gene Modification in Embryonic Stem Cells by Single-Stranded DNA Oligonucleotides

2009 ◽  
pp. 79-99 ◽  
Author(s):  
Marieke Aarts ◽  
Marleen Dekker ◽  
Rob Dekker ◽  
Sandra de Vries ◽  
Anja van der Wal ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Embryonic Stem ◽  
Gene Modification ◽  
Single Stranded Dna ◽  
Dna Oligonucleotides

scholarly journals LNA modification of single-stranded DNA oligonucleotides allows subtle gene modification in mismatch-repair-proficient cells

2016 ◽  
Vol 113 (15) ◽  
pp. 4122-4127 ◽  
Author(s):  
Thomas W. van Ravesteyn ◽  
Marleen Dekker ◽  
Alexander Fish ◽  
Titia K. Sixma ◽  
Astrid Wolters ◽  
...  
Keyword(s):  
Mismatch Repair ◽  
Embryonic Stem ◽  
Gene Modification ◽  
Single Stranded Dna ◽  
Dna Mismatch ◽  
Dna Oligonucleotides ◽  
Single Base Pair Substitution ◽  
Genetic Modifications ◽  
Single Base Pair

Synthetic single-stranded DNA oligonucleotides (ssODNs) can be used to generate subtle genetic modifications in eukaryotic and prokaryotic cells without the requirement for prior generation of DNA double-stranded breaks. However, DNA mismatch repair (MMR) suppresses the efficiency of gene modification by >100-fold. Here we present a commercially available ssODN design that evades MMR and enables subtle gene modification in MMR-proficient cells. The presence of locked nucleic acids (LNAs) in the ssODNs at mismatching bases, or also at directly adjacent bases, allowed 1-, 2-, or 3-bp substitutions in MMR-proficient mouse embryonic stem cells as effectively as in MMR-deficient cells. Additionally, in MMR-proficient Escherichia coli, LNA modification of the ssODNs enabled effective single-base-pair substitution. In vitro, LNA modification of mismatches precluded binding of purified E. coli MMR protein MutS. These findings make ssODN-directed gene modification particularly well suited for applications that require the evaluation of a large number of sequence variants with an easy selectable phenotype.

Characterization of MSH2 variants by endogenous gene modification in mouse embryonic stem cells

2011 ◽  
Vol 32 (4) ◽  
pp. 389-396 ◽  
Author(s):  
Eva A.L. Wielders ◽  
Rob J. Dekker ◽  
Ian Holt ◽  
Glenn E. Morris ◽  
Hein te Riele
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Embryonic Stem ◽  
Mouse Embryonic Stem Cells ◽  
Gene Modification ◽  
Endogenous Gene
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