title: Multiplex ligation-dependent probe amplification for identification of correctly targeted murine embryonic stem cell clones.
abstract: Following locus-specific genome editing of mouse embryonic stem cells (ESCs), the identification of correctly targeted clones remains a challenge. We applied multiplex ligation-dependent probe amplification (MLPA) to screen for homologous recombination-based genomic integration of a knockout construct in which part of a gene is deleted. All candidate ESCs thereby identified were subsequently validated by conventional methods. Thus, MLPA represents a highly reliable as well as cost- and time-efficient alternative to currently applied methods such as Southern blotting and polymerase chain reaction (PCR)-based approaches. It is also applicable to knockin recombination strategies and compatible with the CRISPR/Cas9 system and other genome editing strategies.
authors.affiliation: Department of Clinical Chemistry and Laboratory Diagnostics, Jena University Hospital, 07747 Jena, Germany; Department of Genetics, Institute of Psychiatry and Neurology, 02-957 Warsaw, Poland.
authors.forename: Ewelina
authors.initials: E
authors.lastname: Elert-Dobkowska
authors.affiliation: Institute of Human Genetics, Jena University Hospital, 07747 Jena, Germany.
authors.forename: J Christopher
authors.initials: JC
authors.lastname: Hennings
authors.affiliation: Institute of Human Genetics, Jena University Hospital, 07747 Jena, Germany.
authors.forename: Christian A
authors.initials: CA
authors.lastname: Hübner
authors.affiliation: Department of Clinical Chemistry and Laboratory Diagnostics, Jena University Hospital, 07747 Jena, Germany. Electronic address: christian.beetz@med.uni-jena.de.
authors.forename: Christian
authors.initials: C
authors.lastname: Beetz
mesh_terms: D000818:Animals; D002999:Clone Cells; D018274:Electroporation; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000378:metabolism; D059765:Homologous Recombination / Q000235:genetics; D008565:Membrane Proteins / Q000172:deficiency / Q000378:metabolism; D018345:Mice, Knockout; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*
chemical_list: D008565:Membrane Proteins; C553215:REEP2 protein, mouse
keywords: Embryonic stem cells; Genome engineering; Knockout; MLPA; Mouse model