Figure 1.
Exosc3 expression in the hematopoietic system is required for hematopoietic progenitor activity and embryogenesis. (A) Crystal structure and model of the human EC bound by a synthetic 16 base pair RNA molecule.19 (B) Exosc3 conditional ablation strategy with Cre recombinase expression controlled by the hematopoietic promoter Vav1. Cre recognizes the loxP and lox2372 sites within Exosc3 and promotes inversion, followed by deletion. A GFP cassette is activated after inversion and serves as a recombination marker. Forward (F) and reverse (R) primer sets were used to distinguish wild-type (WT) from conditional by inversion (COIN = C) allele. F and R-inverted (R INV) primer set was used to detect the inverted C allele in hematopoietic tissue. Primer alignment is depicted as black and gray boxes. (C) PCR-based genotyping of WT (expected size of 300 base pair) and Exosc3 COIN allele (expected size of 400 base pair) and amplification of inverted allele (expected size of 500 base pair) specific to hematopoietic tissue. gDNA was obtained from tail clip of Exosc3WT/WT Cre−, Exosc3C/C, and Exosc3C/WT mice for the first 3 lanes. gDNA was obtained from tail clip and fetal liver (FL) of Exosc3C/C Cre+ embryos (2 biological reps) for the 4 lanes below F + R INV. Tail gDNA was used as a negative control for the F + R INV PCR reaction. (D) Embryo genotypes at the indicated developmental stages. The χ-square P values refer to Exosc3C/C Cre+ in comparison with control Exosc3C/C Cre−. (E) Representative E14.5 Exosc3C/C Cre− and Exosc3C/C Cre+ embryos. (F) Fetal liver cell enumeration (13 independent experiments: Exosc3C/C Cre− = 14, Exosc3C/WT Cre− = 33, Exosc3C/WT Cre+ = 28, and Exosc3C/C Cre+ = 14). (G) E14.5 fetal liver cells were plated in a CFU media at 20 000 cells per mL (5 independent experiments: Exosc3C/wt Cre− = 13, Exosc3C/wt Cre+ = 7, Exosc3C/C Cre− = 10 and Exosc3C/C Cre+ = 10). Quantitative data are presented as box and whisker plots, with bounds from the 25th to 75th percentiles, the median line, and whiskers ranging from minimum to maximum values. ∗P < .05, ∗∗P < .01, and ∗∗∗P < .001, by Tukey multiple comparisons test. † indicates nonspecific band; adj, adjusted.

Exosc3 expression in the hematopoietic system is required for hematopoietic progenitor activity and embryogenesis. (A) Crystal structure and model of the human EC bound by a synthetic 16 base pair RNA molecule.19 (B) Exosc3 conditional ablation strategy with Cre recombinase expression controlled by the hematopoietic promoter Vav1. Cre recognizes the loxP and lox2372 sites within Exosc3 and promotes inversion, followed by deletion. A GFP cassette is activated after inversion and serves as a recombination marker. Forward (F) and reverse (R) primer sets were used to distinguish wild-type (WT) from conditional by inversion (COIN = C) allele. F and R-inverted (R INV) primer set was used to detect the inverted C allele in hematopoietic tissue. Primer alignment is depicted as black and gray boxes. (C) PCR-based genotyping of WT (expected size of 300 base pair) and Exosc3 COIN allele (expected size of 400 base pair) and amplification of inverted allele (expected size of 500 base pair) specific to hematopoietic tissue. gDNA was obtained from tail clip of Exosc3WT/WT Cre−, Exosc3C/C, and Exosc3C/WT mice for the first 3 lanes. gDNA was obtained from tail clip and fetal liver (FL) of Exosc3C/C Cre+ embryos (2 biological reps) for the 4 lanes below F + R INV. Tail gDNA was used as a negative control for the F + R INV PCR reaction. (D) Embryo genotypes at the indicated developmental stages. The χ-square P values refer to Exosc3C/C Cre+ in comparison with control Exosc3C/C Cre−. (E) Representative E14.5 Exosc3C/C Cre− and Exosc3C/C Cre+ embryos. (F) Fetal liver cell enumeration (13 independent experiments: Exosc3C/C Cre− = 14, Exosc3C/WT Cre− = 33, Exosc3C/WT Cre+ = 28, and Exosc3C/C Cre+ = 14). (G) E14.5 fetal liver cells were plated in a CFU media at 20 000 cells per mL (5 independent experiments: Exosc3C/wt Cre− = 13, Exosc3C/wt Cre+ = 7, Exosc3C/C Cre− = 10 and Exosc3C/C Cre+ = 10). Quantitative data are presented as box and whisker plots, with bounds from the 25th to 75th percentiles, the median line, and whiskers ranging from minimum to maximum values. ∗P < .05, ∗∗P < .01, and ∗∗∗P < .001, by Tukey multiple comparisons test. † indicates nonspecific band; adj, adjusted.

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