Fig. 2

Generation and characterization of zebrafish transgenic Gmdm2-liver lines. A: Transgenic construct (LF2.8-GFP::Mdm2) of the engineering of transgene GFP::Mdm2, which is GFP fused with zebrafish Mdm2 (GeneBank accession no: NM_131364) into the zebrafish genome. The Not I/Sfi I fragment consists of the zebrafish L-FABP gene 2.8-kb promoter (LF2.8-pro) (Her et al.,[2003a]), transgene GFP::Mdm2, and the SV40 poly(A) signal (SV40 pA). B: Genomic PCR analysis of the production of transgenic zebrafish possessing the transgene GFP::Mdm2. The PCR primer pair P1-P2 was used to identify transgenic zebrafish during breeding. Three transgenic F0 founder lines (Gmdm2-liver#1, Gmdm2-liver#2, and Gmdm2-liver#3) were generated. pLF2.8-GFP::Mdm2 (C+) and WT/G-liver genomic DNA were used as positive and negative controls, respectively. C: Three different cycles of RT-PCR showing the amount of total GFP::Mdm2 and Mdm2 transcripts in hepatic tissues (hepatocytes) of stable transgenic Gmdm2-liver#1, Gmdm2-liver#2, G-liver lines (Her et al.,[2003a]), and wild-type zebrafish. β-actin transcripts are the internal control. D: Immunoblot analysis of GFP:mdm2 expression in the livers of Gmdm2-liver#1 and #2 transgenic zebrafish. An ectopic 60-kDa GFP:mdm2 fusion protein was expressed exclusively in the hepatic tissues of Gmdm2-liver#1 and #2 transgenic zebrafish; higher levels of this protein were expressed exclusively in hepatic tissues of Gmdm-liver#2. Hepatic lysates were isolated from 4-month-old G-liver, WT (wild-type), and Gmdm2-liver#1, and 2 transgenic zebrafish. Hepatic lysates from the G-liver and WT were used as positive and negative controls, respectively. A monoclonal anti-GFP antibody was used for detecting both GFP::Mdm2 and GFP. The same filter was probed with a monoclonal anti-β-actin antibody to calibrate the amount of protein extracts loaded in each lane.