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A VATER-Type anomaly in a stillborn cadaver.
A stillborn male of 29 weeks gestation was macroscopically normal except for a right-sided abdominal mass. The fetus had a large neurenteric cyst. Abdominal ultrasound revealed a massive cystic mass and polyhydramnios. At necropsy, a cystic mass, measuring 12 x 9 x 8 cm, was seen extending from the pelvic to the thoracic cavity. On the right side of the lesion, a vermiform shaped vascular structure was present. Microscopically, the lesion consisted of a large cystic mass containing a fine vascular structure. The patient showed a complex vertebral malformation that resembled the VATER-syndrome.Gene targeting in medaka.
In recent years, the freshwater fish medaka has become a popular model organism for studies on vertebrate development, genetics, and human disease. Historically, it was used to study both embryogenesis and sex determination. This has been greatly facilitated by the availability of transgenic medaka lines and established gene knockout strategies. However, there are still significant technical hurdles in using the medaka as a model system for gene inactivation. Here we present a method for generating targeted mutations in medaka by single-stranded DNA homologous recombination using the CRISPR/Cas9 system. The Cas9 enzyme from the bacterium Streptococcus pyogenes can be guided by single-guide RNAs (sgRNAs) to make a double-strand break in the targeted genomic region, and the sgRNA is designed to be complementary to the selected genomic locus. We show that, if homology-directed repair is efficient, the CRISPR/Cas9 system can efficiently induce mutations in medaka, including large deletions, small in-frame insertions, and more than 15 types of point mutations. In addition, we demonstrate successful knock-in of fluorescent proteins in medaka using the CRISPR/Cas9 system, and we present a detailed protocol for de novo construct design and mutagenesis. This method will be useful for generating a wide range of medaka mutant lines that will be instrumental in furthering our knowledge of vertebrate development, genetics, and human disease.Jim Christian era

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