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Figure 2


Fig. 2. Transgene expression in bi-transgenic mice. (A) RT-PCR of dorsal/ventral skin from bi-transgenic mice and controls using a human LA and LAdel150-specific assay. Presence and absence of transgene is indicated with + and –, respectively. Lanes 1 and 2 are RT-PCR from cell line of HGPS parent AG03504 and HGPS AG03506, respectively. Lane 14 contains cDNA from ventral skin of FVB/N wild-type mice. Lanes 3, 12 and 13 are no-RT control samples for AG03506, tetop-LAG608G+; K5tTA+ and tetop-LAwt+; K5tTA+, respectively. Lane 15 is a no-template control. M, 100 bp ladder (Invitrogen). RT-PCR for TATA-binding protein (TBP) served as a control for the RT (bottom panel). (B,C) Western blot on protein extracts from dorsal/ventral skin of bi-transgenic animals and controls. (B) Filters were simultaneously incubated with antibodies for detection of human lamin A/C (mab3211) and β-actin. Separate western blots, including the same amount of protein extract, were run for detection of green fluorescent protein (GFP, ab290). (C) Enhanced protein separation western blots. An antibody to the N-terminal region of lamin A/C detects lamin A/C of both human and mouse origin, sc-6215 (left). sc-6214 (right) detects prelamin A. (B,C) Same filters were incubated with an antibody to β-actin. F1 line and PCR genotype are indicated above each lane. + and – indicate presence of LA minigene or K5tTA, and absence of K5tTA, respectively. Lane 1 contains protein extracts from HGPS cell line AG03506. Lane 8 is protein extract from a FVB/N wild-type mouse.





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