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

Authors:
Wang J, Li T, Zhou M, Hu Z, Zhou X, Zhou S, Wang N, Huang L, Zhao L, Cao Y, Xiao M, Ma D, Zhou P, Shang Z, Zhou J
In:
Source: Scientific Reports
Publication Date: (2015)
Issue: 5:18454: 10.1038
Research Area:
Cancer Research/Cell Biology
Cells used in publication:
K-562
Species: human
Tissue Origin: blood
Jurkat
Species: human
Tissue Origin: blood
OCI-AML3
Species: human
Tissue Origin: blood
K-11
Species: mouse
Tissue Origin: blood
Culture Media:
Platform:
Nucleofector™ I/II/2b
Experiment
For transient transfection, the TALEN expression plasmids (6?µg per TALEN) were nucleo-transfected into 1?×?106 K562, OCI-AML3 or Jurkat cells using Amaxa nucleofector II and solution V (Lonza) applying program T16, solution T (Lonza) applying program X01 or solution V applying program X01, respectively. After incubation at 30?°C for 48?hours following transfection43, the cells were seeded in 96-well plates at limited dilution (average of 0.4 cells per well). Each clone was cultured for two to three weeks, and a portion of the cells in each well was collected for PCR analysis to detect NHEJ-mediated insertion or deletion
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.