Double Transgenic Reporter Research Articles

Determining the optimal expression method for dual-color imaging

BackgroundFluorescence imaging is a widely used technique that permits for cell-type-specific recording from hundreds of neurons simultaneously. Often, to obtain cell-type-specific recordings from more than one cell type, researchers add an additional fluorescent protein to mark a second neuronal subpopulation. Currently, however, no consensus exists on the best expression method for multiple fluorescent proteins. New MethodWe optimized the coexpression of two fluorescent proteins across multiple brain regions and mouse lines. ResultsThe single-virus method, a viral injection in a double transgenic reporter mouse, results in limited fluorescent coexpression. In contrast the double-virus method, injecting a mixture of two viruses in a Cre driver mouse, results in up to 70 % coexpression of the fluorescent markers in vitro. Using the double-virus method allows for population activity recording and neuronal subpopulation determination. Comparison with Existing MethodThe standard for expressing two fluorescent proteins is to use a double transgenic reporter mouse with a single viral injection. Injecting two viruses into a Cre driver mouse resulted in significantly higher coexpression compared to the standard method. This result generalized to multiple brain regions and mouse lines in vitro, as well as in vivo. ConclusionEfficiently coexpressing multiple fluorescent proteins provides population activity while identifying a neuronal subpopulation of interest. The improved coexpression is applicable to a wide breadth of experiments, ranging from engram investigation to voltage imaging.

VGAT and VGLUT2 expression in MCH and orexin neurons in double transgenic reporter mice

The neuropeptides orexin and melanin-concentrating hormone (MCH), as well as the neurotransmitters GABA (γ-Aminobutyric acid) and glutamate are chief modulators of the sleep-wake states in the posterior hypothalamus. To investigate co-expression of vesicular GABA transporter (VGAT, a marker of GABA neurons) and the vesicular glutamate transporter-2 (VGLUT2, a marker of glutamate neurons) in orexin and MCH neurons, we generated two transgenic mouse lines. One line selectively expressed the reporter gene EYFP in VGAT+ neurons, whereas the other line expressed reporter gene tdTomato in VGLUT2+ neurons. Co-localization between these genetic reporters and orexin or MCH immunofluorescent tags was determined using 3D computer reconstructions of Z stacks that were acquired using a multiphoton laser confocal microscope. Our results demonstrated that MCH neurons expressed neither VGAT nor VGLUT2, suggesting MCH neurons are a separate cluster of cells from VGAT+ GABAergic neurons and VGLUT2+ glutamatergic neurons. Moreover, most orexin neurons expressed VGLUT2, indicating these neurons are glutamatergic. Our data suggested that in the posterior hypothalamus there are four major distinct groups of neurons: VGAT+, orexin+/VGLUT2+, orexin-/VGLUT2+, and MCH neurons. This study facilitated our understanding of the role of these neurotransmitters and neuropeptides in relation to sleep/wake regulation.

The zebrafish operculum: A powerful system to assess osteogenic bioactivities of molecules with pharmacological and toxicological relevance

Bone disorders affect millions of people worldwide and available therapeutics have a limited efficacy, often presenting undesirable side effects. As such, there is a need for novel molecules with bone anabolic properties. The aim of this work was to establish a rapid, reliable and reproducible method to screen for molecules with osteogenic activities, using the zebrafish operculum to assess bone formation. Exposure parameters were optimized through morphological analysis of the developing operculum of larvae exposed to calcitriol, a molecule with known pro-osteogenic properties. An exposure of 3days initiated at 3days post-fertilization was sufficient to stimulate operculum formation, while not affecting survival or development of the larvae. Dose-dependent pro- and anti-osteogenic effects of calcitriol and cobalt chloride, respectively, demonstrated the sensitivity of the method and the suitability of the operculum system. A double transgenic reporter line expressing fluorescent markers for early and mature osteoblasts was used to gain insights into the effects of calcitriol and cobalt at the cellular level, with osteoblast maturation shown to be stimulated and inhibited, respectively, in the operculum of exposed fish. The zebrafish operculum represents a consistent, robust and rapid screening system for the discovery of novel molecules with osteogenic, anti-osteoporotic or osteotoxic activity.

Outer Segment Formation of Transplanted Photoreceptor Precursor Cells

Transplantation of photoreceptor precursor cells (PPCs) into the retina represents a promising treatment for cell replacement in blinding diseases characterized by photoreceptor loss. In preclinical studies, we and others demonstrated that grafted PPCs integrate into the host outer nuclear layer (ONL) and develop into mature photoreceptors. However, a key feature of light detecting photoreceptors, the outer segment (OS) with natively aligned disc membrane staples, has not been studied in detail following transplantation. Therefore, we used as donor cells PPCs isolated from neonatal double transgenic reporter mice in which OSs are selectively labeled by green fluorescent protein while cell bodies are highlighted by red fluorescent protein. PPCs were enriched using CD73-based magnetic associated cell sorting and subsequently transplanted into either adult wild-type or a model of autosomal-dominant retinal degeneration mice. Three weeks post-transplantation, donor photoreceptors were identified based on fluorescent-reporter expression and OS formation was monitored at light and electron microscopy levels. Donor cells that properly integrated into the host wild-type retina developed OSs with the formation of a connecting cilium and well-aligned disc membrane staples similar to the surrounding native cells of the host. Surprisingly, the majority of not-integrated PPCs that remained in the sub-retinal space also generated native-like OSs in wild-type mice and those affected by retinal degeneration. Moreover, they showed an improved photoreceptor maturation and OS formation by comparison to donor cells located on the vitreous side suggesting that environmental cues influence the PPC differentiation and maturation. We conclude that transplanted PPCs, whether integrated or not into the host ONL, are able to generate the cellular structure for effective light detection, a phenomenon observed in wild-type as well as in degenerated retinas. Given that patients suffering from retinitis pigmentosa lose almost all photoreceptors, our findings are of utmost importance for the development of cell-based therapies.

A comprehensive, non-invasive visualization of primordial germ cell development in mice by the Prdm1-mVenus and Dppa3-ECFP double transgenic reporter

The ability to monitor the development of a given cell lineage in a non-invasive manner by fluorescent markers both in vivo and in vitro provides a great advantage for the analysis of the lineage of interest. To date, a number of transgenic or knock-in mouse strains, in which developing germ cells are marked with fluorescent reporters, have been generated. We here describe a novel double transgenic reporter mouse strain that expresses membrane-targeted Venus (mVenus), a brighter variant of yellow fluorescent protein (YFP), under the control of Prdm1 (Blimp1) regulatory elements and enhanced cyan fluorescent protein (ECFP) under the control of Dppa3 (Stella/Pgc7). The double transgenic strain unambiguously marked Prdm1 expression in the lineage-restricted precursors of primordial germ cells (PGCs) in the proximal epiblast at embryonic day (E) 6.25 and specifically illuminated Prdm1- and Dppa3-positive migrating PGCs after E8.5. The double transgenic reporter also precisely recapitulated dynamic embryonic expression of Prdm1 outside the germ cell lineage. Moreover, we derived ES cells that bore both transgenes. These cells made a robust contribution both to the germ and somatic cell lineages in chimeras with accurate Prdm1-mVenus and Dppa3-ECFP expression. The transgenic strain and the ES cells will serve as valuable experimental materials not only for analyzing the origin and properties of the germ cell lineage in vivo, but also for establishing a culture system to efficiently induce proper germ cells with temporally coordinated Prdm1 and Dppa3 expression in vitro.

Kartagemer's Syndrome in an American Indian Girl: Report of a Case

Bone disorders affect millions of people worldwide and available therapeutics have a limited efficacy, often presenting undesirable side effects. As such, there is a need for novel molecules with bone anabolic properties. The aim of this work was to establish a rapid, reliable and reproducible method to screen for molecules with osteogenic activities, using the zebrafish operculum to assess bone formation. Exposure parameters were optimized through morphological analysis of the developing operculum of larvae exposed to calcitriol, a molecule with known pro-osteogenic properties. An exposure of 3 days initiated at 3 days post-fertilization was sufficient to stimulate operculum formation, while not affecting survival or development of the larvae. Dose-dependent pro- and anti-osteogenic effects of calcitriol and cobalt chloride, respectively, demonstrated the sensitivity of the method and the suitability of the operculum system. A double transgenic reporter line expressing fluorescent markers for early and mature osteoblasts was used to gain insights into the effects of calcitriol and cobalt at the cellular level, with osteoblast maturation shown to be stimulated and inhibited, respectively, in the operculum of exposed fish. The zebrafish operculum represents a consistent, robust and rapid screening system for the discovery of novel molecules with osteogenic, anti-osteoporotic or osteotoxic activity.