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BACKGROUND:Choroid plexus epithelial cells express high levels of transthyretin, produce cerebrospinal fluid and many of its proteins, and make up the blood-cerebrospinal fluid barrier. Choroid plexus epithelial cells are vital to brain health and may be involved in neurological diseases. Transgenic mice containing fluorescent and luminescent reporters of these cells would facilitate their study in health and disease, but prior transgenic reporters lost expression over the early postnatal period. METHODS:Human bacterial artificial chromosomes in which the transthyretin coding sequence was replaced with DNA for tdTomato or luciferase 2 were used in pronuclear injections to produce transgenic mice. These mice were characterized by visualizing red fluorescence, immunostaining, real-time reverse transcription polymerase chain reaction, and luciferase enzyme assay. RESULTS:Reporters were faithfully expressed in cells that express transthyretin constitutively, including choroid plexus epithelial cells, retinal pigment epithelium, pancreatic islets, and liver. Expression of tdTomato in choroid plexus began at the appropriate embryonic age, being detectable by E11.5. Relative levels of tdTomato transcript in the liver and choroid plexus paralleled relative levels of transcripts for transthyretin. Expression remained robust over the first postnatal year, although choroid plexus transcripts of tdTomato declined slightly with age whereas transthyretin remained constant. TdTomato expression patterns were consistent across three founder lines, displayed no sex differences, and were stable across several generations. Two of the tdTomato lines were bred to homozygosity, and homozygous mice are healthy and fertile. The usefulness of tdTomato reporters in visualizing and analyzing live Transwell cultures was demonstrated. Luciferase activity was very high in homogenates of choroid plexus and continued to be expressed through adulthood. Luciferase also was detectable in eye and pancreas. CONCLUSIONS:Transgenic mice bearing fluorescent and luminescent reporters of transthyretin should prove useful for tracking transplanted choroid plexus epithelial cells, for purifying the cells, and for reporting their derivation from stem cells. They also should prove useful for studying transthyretin synthesis by other cell types, as transthyretin has been implicated in many functions and conditions, including clearance of β-amyloid peptides associated with Alzheimer's disease, heat shock in neurons, processing of neuropeptides, nerve regeneration, astrocyte metabolism, and transthyretin amyloidosis.

A general method for efficiently hatching sexually produced diapausing embryos of the microcrustacean Daphnia is valuable for establishing Daphnia as a genetic model system. In this study, we examine the effect of ultraviolet light and different amounts of storage time in darkness on the hatching efficiency in two species of the Daphnia pulex species complex, D. pulex and Daphnia pulicaria. We identified a set of lighting conditions that can trigger 80% to ~100% hatching rate for embryos produced through selfing, outcrossing, and obligate parthenogenesis. Furthermore, we found that a storage time of at least 2 weeks in the dark before exposing embryos to ultraviolet light is critical for achieving high hatching rate. The identification of these key factors for hatching diapausing embryos can greatly facilitate future Daphnia research involving complex breeding designs as well as investigating the genetic switch that activates the hatching of diapausing embryos. Research highlights: This study examines the effect of ultraviolet light on activating diapausing embryos of Daphnia pulex and Daphnia pulicaria. The identified optimal conditions can induce hatching efficiency of 80–100% in different geographical isolates of Daphnia. [ABSTRACT FROM AUTHOR]