Isolation of elaA promoter (1.9 kb) and construction of the chimeric plasmid pElaA-EGFP have been described previously [17 (link)] The liver-specific promoter (2.8 kb) derived from the zebrafish liver fatty acid binding protein gene was provided by Dr. G.-M. Her and was inserted into pDsRed-Express-1 (Clontech, USA) to make the chimeric plasmid pLFABP-RFP. Both plasmids were linearized, mixed with 0.25% phenol red solution (1:1:1) at a final concentration of 100 ng/μl of each plasmid. Microinjection was carried out at the 1–2 cell stage. The DNA solution was injected into the boundary between the yolk and blastodisc. After microinjection, the embryos were maintained in egg water [29 ] with ~0.0005% methylene blue in a 28.5°C incubator. Transgenic founders were screened by observation of F1 embryos for RFP and GFP expression. 444 injected embryos were raised to adult and 66 of them were screened for transgenics. Two of them were found to produce F1 embryos with strong liver-specific RFP expression and exocrine pancreas-specific GFP expression. Thus, two stable transgenic lines were established and both showed standard Mendelian inheritance from F2 generation onwards. Since identical reporter gene expression patterns were observed in the two lines, only one line, named LiPan, was used for further characterization. Homozygous LiPan zebrafish were viable and had no visible phenotype. The LiPan line has been maintained in our laboratories for over eight generations and co-expression of RFP in the liver and GFP in the exocrine pancreas is always observed. Thus the two injected DNA constructs are likely co-integrated into the same chromosomal locus.
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