Fig. 4

Transgenic zebrafish allows for visualization of human APOA-I in vivo. Live tg(lfabp10:hAPOA-I-mCherry) (A–C) and tg(ifabp:hAPOA-I-mCherry) (E–F) larvae show hAPOA-I-mCherry accumulation in the liver and intestine. hAPOA-I-mCherry derived from the liver (outlined by dotted line) accumulates in the intestine outlined by wavy line) and hAPOA-I-mCherry derived from the intestine accumulates in the liver (B and C). Tissue specificity of promoters used in APOA-I-mCherry transgenic fish revealed using whole mount in situ hybridization with antisense riboprobes to mCherry mRNA (D and G). No expression was detected with the sense probes (n = 10–12 fish); all larvae 6-dpf. ApoA-I-mCherry fusion protein is made in transgenic zebrafish and partially degraded (H). ApoA-I-mCherry fusion protein (arrow head) is made in transgenic zebrafish as expected size (50 kDa) (representative image from three experiments). mCherry antisera detected two different bands from Tg(lfabp:hApoA-I-mcherry) and Tg(ifabp:hApoA-I-mcherry) (I). The higher molecular weight bands (arrow head) represent ApoA-I-mCherry full-length protein. The lower molecular weight bands (arrow) is possibly a degradation product of similar size as mCherry protein made from Tg(ef1a:mcherry-CVLL) (representative image from two experiments). mCherry flourscence on native gel (representative image from four experiments) (J). Immunofluorescence for endogenous zApoA-Ia and zApoA-Ib in wild-type fish show punctae within the intestine similar to hAPOA-I-mCherry accumulation (representative image from three experiments) (K). APOA-I, apolipoprotein A-I; dpf, days postfertilization; E, Tg(ef1a:mcherry-CVLL); hAPOA-I, human APOA-I; I, Tg(ifabp:hApoA-I-mcherry); L, Tg(lfabp:hApoA-I-mcherry); W, wild-type; zAPOA-1, zebrafish APOA-1.