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Sci Rep. 2014 Aug 13;4:6048. doi: 10.1038/srep06048.

Using RNA-seq and targeted nucleases to identify mechanisms of drug resistance in acute myeloid leukemia.

Author information

1
Masonic Cancer Center, University of Minnesota, Minneapolis, MN, USA.
2
1] Masonic Cancer Center, University of Minnesota, Minneapolis, MN, USA [2] Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN, USA [3] Center for Genome Engineering, University of Minnesota, Minneapolis, MN, USA.
3
1] Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN, USA [2] Center for Genome Engineering, University of Minnesota, Minneapolis, MN, USA.
4
1] Masonic Cancer Center, University of Minnesota, Minneapolis, MN, USA [2] Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN, USA [3] Center for Genome Engineering, University of Minnesota, Minneapolis, MN, USA [4] Brain Tumor Program, University of Minnesota, Minneapolis, MN, USA [5] Department of Pediatrics, University of Minnesota, Minneapolis, MN, USA.

Abstract

The evolution from microarrays to transcriptome deep-sequencing (RNA-seq) and from RNA interference to gene knockouts using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) and Transcription Activator-Like Effector Nucleases (TALENs) has provided a new experimental partnership for identifying and quantifying the effects of gene changes on drug resistance. Here we describe the results from deep-sequencing of RNA derived from two cytarabine (Ara-C) resistance acute myeloid leukemia (AML) cell lines, and present CRISPR and TALEN based methods for accomplishing complete gene knockout (KO) in AML cells. We found protein modifying loss-of-function mutations in Dck in both Ara-C resistant cell lines. CRISPR and TALEN-based KO of Dck dramatically increased the IC₅₀ of Ara-C and introduction of a DCK overexpression vector into Dck KO clones resulted in a significant increase in Ara-C sensitivity. This effort demonstrates the power of using transcriptome analysis and CRISPR/TALEN-based KOs to identify and verify genes associated with drug resistance.

PMID:
25116387
PMCID:
PMC4131221
DOI:
10.1038/srep06048
[Indexed for MEDLINE]
Free PMC Article

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