title: Simple and efficient methods for enrichment and isolation of endonuclease modified cells.
abstract: The advent of Transcription Activator-Like Effector Nucleases (TALENs), and similar technologies such as CRISPR, provide a straightforward and cost effective option for targeted gene knockout (KO). Yet, there is still a need for methods that allow for enrichment and isolation of modified cells for genetic studies and therapeutics based on gene modified human cells. We have developed and validated two methods for simple enrichment and isolation of single or multiplex gene KO's in transformed, immortalized, and human progenitor cells. These methods rely on selection of a phenotypic change such as resistance to a particular drug or ability to grow in a selective environment. The first method, termed co-transposition, utilizes integration of a piggyBac transposon vector encoding a drug resistance gene. The second method, termed co-targeting, utilizes TALENs to KO any gene that when lost induces a selectable phenotype. Using these methods we also show removal of entire genes and demonstrate that TALENs function in human CD34+ progenitor cells. Further, co-transposition can be used to generate conditional KO cell lines utilizing an inducible cDNA rescue transposon vector. These methods allow for robust enrichment and isolation of KO cells in a rapid and efficient manner.
authors.affiliation: Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, Minnesota, United States of America; Center for Genome Engineering and Institute of Human Genetics, University of Minnesota, Minneapolis, Minnesota, United States of America; Masonic Cancer Center, University of Minnesota, Minneapolis, Minnesota, United States of America.
authors.forename: Branden S
authors.initials: BS
authors.lastname: Moriarity
authors.affiliation: Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, Minnesota, United States of America; Center for Genome Engineering and Institute of Human Genetics, University of Minnesota, Minneapolis, Minnesota, United States of America; Masonic Cancer Center, University of Minnesota, Minneapolis, Minnesota, United States of America.
authors.forename: Eric P
authors.initials: EP
authors.lastname: Rahrmann
authors.affiliation: Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, Minnesota, United States of America; Center for Genome Engineering and Institute of Human Genetics, University of Minnesota, Minneapolis, Minnesota, United States of America; Masonic Cancer Center, University of Minnesota, Minneapolis, Minnesota, United States of America.
authors.forename: Dominic A
authors.initials: DA
authors.lastname: Beckmann
authors.affiliation: Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, Minnesota, United States of America; Masonic Cancer Center, University of Minnesota, Minneapolis, Minnesota, United States of America.
authors.forename: Caitlin B
authors.initials: CB
authors.lastname: Conboy
authors.affiliation: Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, Minnesota, United States of America; Masonic Cancer Center, University of Minnesota, Minneapolis, Minnesota, United States of America.
authors.forename: Adrienne L
authors.initials: AL
authors.lastname: Watson
authors.affiliation: Center for Genome Engineering and Institute of Human Genetics, University of Minnesota, Minneapolis, Minnesota, United States of America; Department of Animal Science, University of Minnesota, Minneapolis, Minnesota, United States of America; Recombinetics, Inc., Saint Paul, Minnesota, United States of America.
authors.forename: Daniel F
authors.initials: DF
authors.lastname: Carlson
authors.affiliation: Discovery Genomics, Inc, Minneapolis, Minnesota, United States of America.
authors.forename: Erik R
authors.initials: ER
authors.lastname: Olson
authors.affiliation: Discovery Genomics, Inc, Minneapolis, Minnesota, United States of America.
authors.forename: Kendra A
authors.initials: KA
authors.lastname: Hyland
authors.affiliation: Center for Genome Engineering and Institute of Human Genetics, University of Minnesota, Minneapolis, Minnesota, United States of America; Department of Animal Science, University of Minnesota, Minneapolis, Minnesota, United States of America; Recombinetics, Inc., Saint Paul, Minnesota, United States of America.
authors.forename: Scott C
authors.initials: SC
authors.lastname: Fahrenkrug
authors.affiliation: Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, Minnesota, United States of America; Center for Genome Engineering and Institute of Human Genetics, University of Minnesota, Minneapolis, Minnesota, United States of America; Discovery Genomics, Inc, Minneapolis, Minnesota, United States of America.
authors.forename: R Scott
authors.initials: RS
authors.lastname: McIvor
authors.affiliation: Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, Minnesota, United States of America; Center for Genome Engineering and Institute of Human Genetics, University of Minnesota, Minneapolis, Minnesota, United States of America; Masonic Cancer Center, University of Minnesota, Minneapolis, Minnesota, United States of America; Discovery Genomics, Inc, Minneapolis, Minnesota, United States of America; Department of Pediatrics, University of Minnesota, Minneapolis, Minnesota, United States of America.
authors.forename: David A
authors.initials: DA
authors.lastname: Largaespada
mesh_terms: D002460:Cell Line; D002469:Cell Separation / Q000379:methods*; D004251:DNA Transposable Elements; D004720:Endonucleases / Q000096:biosynthesis / Q000235:genetics; D055786:Gene Knockout Techniques / Q000379:methods*; D005822:Genetic Vectors; D006801:Humans; D013234:Stem Cells / Q000166:cytology / Q000378:metabolism
chemical_list: D004251:DNA Transposable Elements; D004720:Endonucleases