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. 2007 Apr 26;104(19):8053–8058. doi: 10.1073/pnas.0611669104

Fig. 1.

Fig. 1.

Proliferation and tumorigenic properties of gene-trapped astrocytes derived from the B8 GEM astrocytoma model. (A) MTT proliferation assays of NMAs from CD1-ICR newborn pups (NMA-P0), newborn B8 pups (B8-P0), both transduced with empty vector controls (Neg), gene-trapped B8-P0 astrocyte clones (GT1, GT2, and GT3), and B8–3mth astrocytoma cultures derived from a mouse harboring a HGA. Gene-trapped B8-P0 clones have a proliferation advantage compared with parental and Neg NMA-P0 and B8-P0 astrocytes, approaching the proliferation rate of the transformed B8–3mth astrocytoma cultures. (B Upper) Pathological features of invasive malignant astrocytoma in the frontal cortex of Nod-Scid mice 1 month after injection of 106 B8–3mth astrocytes (H&E). β-Gal staining confirms expression of the GFAP:V12HaRas-IRES-LacZ transgene. GATA6 expression is lost in the HGA. (Lower) Pathological features of invasive malignant astrocytoma in the frontal cortex of Nod-Scid mice that developed in ≈7% of B8-P0 gene-trapped astrocytes (GT1 clone shown; H&E), with β-gal expression from both the GFAP:V12HaRas-IRES-LacZ transgene and the gene-trap vector. Parental or empty gene-trap vector-negative NMA and B8-P0 controls did not grow in Nod-Scid mice up to 6 months of observation. The finding of loss of Gata6 expression in the HGA suggests that an acquired mutation occurred in the nontargeted gene-trapped allele. (C) B8 GEM astrocytomas at the age of 1 month (LGA, Upper) and 3 months (HGA, Lower) (H&E). Gata6 is expressed in 1-month LGA (Upper Right) but absent in 3-month HGA (Lower Right), suggesting a role in tumor progression. β-Gal staining (Center) confirms expression of the GFAP:V12HaRas-IRES-LacZ transgene in the transformed astrocytes. GATA6 is abundantly expressed in wild-type age-matched brains (13). (Magnifications: ×400 for B and C and ×40 for Insets in B Left. Scale bars: 25 μm for B and C and 250 μm for Insets in B Left.) (D) Western blot analysis demonstrating that p19ARF and p53 expression is relatively unchanged in the B8 gene-trapped clones (GT1, GT2, and GT3) when compared with the B8-P0 parental cells. p19ARF expression is absent in the B8–3mth astrocytoma cells. Approximately 40 μg of protein lysates was loaded in each well. GAPDH was used to assess the amount of protein loaded.