The retroviral insertion in the scarb2a gene from zebrafish. A: Diagram of the scarb2a exon-intron structure and the location of the retroviral insertion at the first intron. Scarb2a protein is represented by a box in which the predicted positions of the two transmembrane domains (TM) and the predicted Asn residues for N-glycosylation sites are indicated by dotted lines. B: Phylogenetic tree using the zebrafish Scarb1 and CD36 sequences as an outgroup. The tree was built with MacVector 12.0 using “Neighbor joining” as the tree-building method in the Best tree mode with uncorrected “p” distance with the gaps distributed proportionally. Numbers above lines indicate substitutions per site. D.r. (Danio rerio “zebrafish”), C.i. (Ciona intestinalis), T.n. (Tetraodon nigroviridis “pufferfish”), X.t. (Xenopus tropicalis), M.d. (Monodelphis domestica “opossum”) and H.s. (Homo sapiens). C: Simultaneous DNA and total RNA purification from 20 phenotypic scarb2ahi1463Tg mutant embryos allowed us to determine that all tested embryos carried the mutagenic insertion in the scarb2a gene and at the same time were null for scarb2a because they could not express scarb2a mRNA. This was performed by genotyping by PCR and by RT-PCR amplification, respectively. D: Genotyping was conducted using three primers, two flanking the insertion (1463c and 1463c1) and one at the retroviral insertion site (MSL4) but oriented toward the 52-end primer. Approximately 80 adults raised from an outcross were fin clipped and genotyped, and then only the identified carriers (approximately 50%) were used to obtain homozygous scarb2ahi1463Tg embryos. E: Whole-mount in situ hybridization to detect the expression of scarb2a mRNA in 24 hpf embryos. scarb2a mRNA was not detected in approximately 1/4 of the embryos. In the other 3/4 of the embryos, the scarb2a RNA antisense probe was detected in the brain and the posterior notochord (see red arrows).
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