TALENs-mediated gene disruption of FLT3 in leukemia cells: Using genome-editing approach for exploring the molecular basis of gene abnormality.

Jue Wang,Liang Huang,Na Wang,Shiqiu Zhou,Yang Cao,Mi Zhou,Zheng Hu,Lei Zhao,Ding Ma,Xiaoxi Zhou,Tongjuan Li,Min Xiao,Pengfei Zhou,Jianfeng Zhou,Zhen Shang

doi:10.1038/srep18454

Abstract

Novel analytic tools are needed to elucidate the molecular basis of leukemia-relevant gene mutations in the post-genome era. We generated isogenic leukemia cell clones in which the FLT3 gene was disrupted in a single allele using TALENs. Isogenic clones with mono-allelic disrupted FLT3 were compared to an isogenic wild-type control clone and parental leukemia cells for transcriptional expression, downstream FLT3 signaling and proliferation capacity. The global gene expression profiles of mutant K562 clones and corresponding wild-type controls were compared using RNA-seq. The transcriptional levels and the ligand-dependent autophosphorylation of FLT3 were decreased in the mutant clones. TALENs-mediated FLT3 haplo-insufficiency impaired cell proliferation and colony formation in vitro. These inhibitory effects were maintained in vivo, improving the survival of NOD/SCID mice transplanted with mutant K562 clones. Cluster analysis revealed that the gene expression pattern of isogenic clones was determined by the FLT3 mutant status rather than the deviation among individual isogenic clones. Differentially expressed genes between the mutant and wild-type clones revealed an activation of nonsense-mediated decay pathway in mutant K562 clones as well as an inhibited FLT3 signaling. Our data support that this genome-editing approach is a robust and generally applicable platform to explore the molecular bases of gene mutations.

Highlights

The TALEN target site was selected within the sequence of exon[14], which encodes the JM domain and is located upstream of the tyrosine kinase domains (TKDs) and the kinase insert (KI) domain, such that insertions or deletions caused by non-homologous end joining (NHEJ) could result in disruption of the reading frame or the formation of a premature stop codon
Using a designed TALENs approach, we generated a panel of isogenic clones carrying a mono-allelic mutation of FMS-like tyrosine kinase 3 (FLT3) in K562 and OCI-AML3 leukemia cells
We hypothesized that the resulting isogenic clones, which theoretically carried the same genetic background except for the FLT3 mutant status, serve as a cellular model to characterize the biological effects and critical molecular events caused by TALEN-mediated disruption of the FLT3 gene

Summary

Introduction

Genetically engineered animal models will remain among the most reliable tools to study the association between genetic defects and clinical phenotypes, and contribution to the design and development of novel molecular-targeted strategies. Novel analytic tools are urgently needed to address the molecular mechanisms underlying leukemia-relevant gene function in the post-genome era. Transcription activator-like effector (TALE) nucleases (TALENs), an efficient genome editing tool, are artificial fusion proteins containing the catalytic domain of the endonuclease FokI and a designed TALE DNA-binding domain that recognizes a specific DNA sequence[7,8]. We generated isogenic clones, in two individual leukemia cell lines, by disrupting FMS-like tyrosine kinase 3 (FLT3) gene in a single allele using designed TALENs. The resulting isogenic clones which were only different in the FLT3 mutant status were compared for FLT3 downstream signaling, proliferation capacity and transcriptional expression. Our data strongly support that this genome-editing approach can serve as a robust and generally applicable platform for exploring the molecular basis of a given gene abnormality

Methods

Results

Conclusion