Figure 1. Bub1 transgenic mice overexpress Bub1 protein in a wide variety of tissues and cell types. (A) Overview of the approach used to generate Bub1 transgenic mouse strains. (Top) Transgenic mice were generated in which the HA-Bub1 and EGFP transgenes are inactive due to the presence of a floxed β-geo “STOP” cassette (consisting of β-galactosidase-neomycin fusion gene and three tandemly arranged polyadenylation sites) immediately downstream of the CAGGS promoter. We bred these transgenics to protamine-Cre transgenic mice (O’Gorman et al., 1997) to excise the STOP cassette in the male germline. (Bottom) Breeding of double-transgenic males to wild-type females, yielded offspring in which the CAGGS promoter was juxtaposed with the HA-Bub1 and EGFP coding regions in all cells. IRES, internal ribosomal entry site; PA, protamine. (B) Immunoblot analysis of mitotic and asynchronous lysates from transgenic and wild-type primary MEFs. Blots were probed for endogenous Bub1 (Bub1), exogenous HA-Bub1 (HA), and GFP (GFP). Actin and pH3 were used as loading controls. (C) QRT-PCR for Bub1 transcripts in cycling MEFs of the indicated genotypes. Orange and black arrows mark primer positions for analysis of total (endogenous and exogenous, p1/p2) and endogenous Bub1 (p3/p4), respectively. Data shown are the mean ± SEM (n = 3 independent cell lines, in triplicate). Values were normalized to TBP. (D) EGFP fluorescence from 1-d-old pups of the indicated genotypes. (E) Representative images of wild-type, Bub1T85, and Bub1T264 MEFs in prometaphase coimmunostained with anti-Bub1 and anti-centromere antibodies. DNA was visualized with Hoechst. Bar, 10 µm. (F) Total Bub1 transcripts in various tissues and cell types from mice of the indicated genotypes (primer pair p1/p2 was used in this qRT-PCR analysis). Data shown are the mean ± SEM (n = 3 mice per genotype, in triplicate). Values were normalized to TBP except bone marrow, which was normalized to GAPDH. (G) Western blot analysis of extracts of the indicated tissues and cell types for Bub1. Ponceau S served as a loading control.
Figure 1.

Bub1 transgenic mice overexpress Bub1 protein in a wide variety of tissues and cell types. (A) Overview of the approach used to generate Bub1 transgenic mouse strains. (Top) Transgenic mice were generated in which the HA-Bub1 and EGFP transgenes are inactive due to the presence of a floxed β-geo “STOP” cassette (consisting of β-galactosidase-neomycin fusion gene and three tandemly arranged polyadenylation sites) immediately downstream of the CAGGS promoter. We bred these transgenics to protamine-Cre transgenic mice (O’Gorman et al., 1997) to excise the STOP cassette in the male germline. (Bottom) Breeding of double-transgenic males to wild-type females, yielded offspring in which the CAGGS promoter was juxtaposed with the HA-Bub1 and EGFP coding regions in all cells. IRES, internal ribosomal entry site; PA, protamine. (B) Immunoblot analysis of mitotic and asynchronous lysates from transgenic and wild-type primary MEFs. Blots were probed for endogenous Bub1 (Bub1), exogenous HA-Bub1 (HA), and GFP (GFP). Actin and pH3 were used as loading controls. (C) QRT-PCR for Bub1 transcripts in cycling MEFs of the indicated genotypes. Orange and black arrows mark primer positions for analysis of total (endogenous and exogenous, p1/p2) and endogenous Bub1 (p3/p4), respectively. Data shown are the mean ± SEM (n = 3 independent cell lines, in triplicate). Values were normalized to TBP. (D) EGFP fluorescence from 1-d-old pups of the indicated genotypes. (E) Representative images of wild-type, Bub1T85, and Bub1T264 MEFs in prometaphase coimmunostained with anti-Bub1 and anti-centromere antibodies. DNA was visualized with Hoechst. Bar, 10 µm. (F) Total Bub1 transcripts in various tissues and cell types from mice of the indicated genotypes (primer pair p1/p2 was used in this qRT-PCR analysis). Data shown are the mean ± SEM (n = 3 mice per genotype, in triplicate). Values were normalized to TBP except bone marrow, which was normalized to GAPDH. (G) Western blot analysis of extracts of the indicated tissues and cell types for Bub1. Ponceau S served as a loading control.

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