FLI1 monoallelic expression combined with its hemizygous loss underlies Paris-Trousseau/Jacobsen thrombopenia
J. Clin. Invest. Hana Raslova, et al. 114:77 doi:10.1172/JCI21197 [
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Figure 4Fli1 expression throughout normal MK differentiation. CD34
+ cells were cultured in the presence of TPO and analyzed at day 6. (
A)
Fli1 expression level determined by real-time RT-PCR. Diploid MKs were sorted according to their differentiation stages.
Fli1 and two housekeeping β
2-M and β
5-tubulin (β
5-T) genes (constant throughout MK differentiation; our unpublished data) were amplified in separate wells in five independent experiments (four using β
2-M and one using β
5-T). The relative expression level of
Fli1 in each fraction was normalized to the corresponding values for β
2-M or β
5-T. A representative experiment is shown with error bars representing the SD of the mean of triplicate wells. (
B) Representative picture of TPO-cultured cells showing the distribution of Fli1 nuclear RNA (green) and chromosome 12 (red) visualized by FISH. Cell with a monoallelic expression of
Fli1 is on the left, and cell with its biallelic expression is on the right.The number of green spots indicates the number of transcribed
Fli1 alleles. The number of
Fli1 alleles in the same MK is determined by the number of red spots. Chromatin is counterstained with DAPI (blue). (
C) Statistical analysis of monoallelic/biallelic expression of
Fli1 in diploid MKs during their differentiation. Cells were sorted as described in
A, and RNA-FISH was performed as described in
B. One hundred cells of each population were analyzed on an epifluorescence microscope (Nikon Eclipse 600) using a ×60 objective for the presence of Fli1 pre-mRNA. The error bars represent the SD of the mean of three independent experiments.
