Abstract
The vitamin E regulatory protein, the alpha-tocopherol transfer protein (Ttpa), is necessary for zebrafish embryo development. To evaluate zebrafish embryo Ttpa function, we generated a fluorescent-tagged zebrafish transgenic line using CRISPR-Cas9 technology. One-cell stage embryos (from Casper (colorless) zebrafish adults) were injected the mScarlet coding sequence in combination with cas9 protein complexed to single guide RNA molecule targeting 5′ of the ttpa genomic region. Embryos were genotyped for proper insertion of the mScarlet coding sequence, raised to adulthood and successively in-crossed to produce the homozygote RedEfish (mScarlet: GSG-T2A: Ttpa). RedEfish were characterized by in vivo fluorescence detection at 1, 7 and 14 days post-fertilization (dpf). Fluorescent color was detectable in RedEfish embryos at 1 dpf; it was distributed throughout the developing brain, posterior tailbud and yolk sac. At 7 dpf, the RedEfish was identifiable by fluorescence in olfactory pits, gill arches, pectoral fins, posterior tail region and residual yolk sac. Subsequently (14 dpf), the mScarlet protein was found in olfactory pits, distributed throughout the digestive tract, along the lateral line and especially in caudal vertebrae. No adverse morphological outcomes or developmental delays were observed. The RedEfish will be a powerful model to study Ttpa function during embryo development.
Highlights
Vitamin E (VitE, α-tocopherol) is necessary for embryonic development [1]
Success was defined as integration of the mScarlet sequence 50 of ttpa without erroneous insertions or deletions (INDELs), as determined by Sanger Sequencing
We present the RedEfish, a new model for studying Ttpa localization and trafficking
Summary
Low maternal VitE in human serum and cord blood is linked to prematurity, low birth weight [2], increased risk of miscarriage [3] and intrauterine growth restriction [4]. Preterm babies were found with lower VitE relative to term babies of mothers with similar cord blood VitE levels [5] and circulating VitE in women at early and late term was positively correlated to fetal growth and decreased risk for low birth weight [6]. TTPA is localized primarily in hepatocytes, where it facilitates VitE trafficking from recycling endosomes to the membrane in exchange for inositol-triphosphate (Ins3P) [9], the Antioxidants 2021, 10, 965. Antioxidants 2021, 10, 965 transfer of VitE to nascent lipoproteins is facilitated by ABCA1 [10], an ATP-dependent transporter. In addition to the liver, TTPA is expressed in the human yolk sac [11]
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