Simple and efficient methods for enrichment and isolation of endonuclease modified cells

Branden S. Moriarity, Eric P. Rahrmann, Dominic A. Beckmann, Caitlin B. Conboy, Adrienne L. Watson, Daniel F. Carlson, Erik R. Olson, Kendra A. Hyland, Scott C. Fahrenkrug, R. Scott McIvor, David A. Largaespada

Research output: Contribution to journalArticle

11 Citations (Scopus)

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.

Original languageEnglish (US)
Article numbere96114
JournalPloS one
Volume9
Issue number5
DOIs
StatePublished - May 5 2014

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Endonucleases
Genes
Transcription
nucleases
cells
Gene Knockout Techniques
transposition (genetics)
transcription (genetics)
Cell Separation
gene targeting
transposons
Clustered Regularly Interspaced Short Palindromic Repeats
methodology
stem cells
Cells
Stem Cells
genes
Pharmaceutical Preparations
drug resistance
Drug Resistance

Cite this

Simple and efficient methods for enrichment and isolation of endonuclease modified cells. / Moriarity, Branden S.; Rahrmann, Eric P.; Beckmann, Dominic A.; Conboy, Caitlin B.; Watson, Adrienne L.; Carlson, Daniel F.; Olson, Erik R.; Hyland, Kendra A.; Fahrenkrug, Scott C.; Scott McIvor, R.; Largaespada, David A.

In: PloS one, Vol. 9, No. 5, e96114, 05.05.2014.

Research output: Contribution to journalArticle

Moriarity, BS, Rahrmann, EP, Beckmann, DA, Conboy, CB, Watson, AL, Carlson, DF, Olson, ER, Hyland, KA, Fahrenkrug, SC, Scott McIvor, R & Largaespada, DA 2014, 'Simple and efficient methods for enrichment and isolation of endonuclease modified cells', PloS one, vol. 9, no. 5, e96114. https://doi.org/10.1371/journal.pone.0096114
Moriarity, Branden S. ; Rahrmann, Eric P. ; Beckmann, Dominic A. ; Conboy, Caitlin B. ; Watson, Adrienne L. ; Carlson, Daniel F. ; Olson, Erik R. ; Hyland, Kendra A. ; Fahrenkrug, Scott C. ; Scott McIvor, R. ; Largaespada, David A. / Simple and efficient methods for enrichment and isolation of endonuclease modified cells. In: PloS one. 2014 ; Vol. 9, No. 5.
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