Fluorescent Embryos Research Articles

Direct Injection of Recombinant AAV-Containing Solution into the Oviductal Lumen of Pregnant Mice Caused In Situ Infection of Both Preimplantation Embryos and Oviductal Epithelium.

Adeno-associated virus (AAV) vector is an efficient viral-based gene delivery tool used with many types of cells and tissues, including neuronal cells and muscles. AAV serotype 6 (AAV-6), one of numerous AAV serotypes, was recently found to efficiently transduce mouse preimplantation embryos. Furthermore, through coupling with a clustered, regularly interspaced, short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system—a modern genome editing technology—AAV-6 has been shown to effectively create a mutation at a target locus, which relies on isolation of zygotes, in vitro viral infection, and transplantation of the infected embryos to recipient females. Unfortunately, this procedure, termed “ex vivo handling of embryos”, requires considerable investment of capital, time, and effort. Direct transduction of preimplantation embryos through the introduction of AAV-6 into the oviductal lumen of pregnant females would be an ideal approach. In this study, we injected various types of recombinant AAV vectors (namely, rAAV-CAG-EGFP-1, -2, -5, and -6, each carrying an enhanced green fluorescent protein [EGFP] cDNA whose expression is under the influence of a cytomegalovirus enhancer + chicken β-actin promoter) into the ampulla region of oviducts in pregnant female mice at Day 0.7 of pregnancy (corresponding to the late 1-cell stage), and EGFP-derived green fluorescence was assessed in the respective morulae. The highest levels of fluorescence were observed in rAAV-CAG-EGFP-6. The oviductal epithelium was distinctly fluorescent. The fluorescence in embryos peaked at the morula stage. Our results indicate that intra-oviductal injection of AAV-6 vectors is the most effective method for transducing zona pellucida-enclosed preimplantation embryos in situ. AAV-6 vectors could be a useful tool in the genetic manipulation of early embryos, as well as oviductal epithelial cells.

Estrogen sensitive liver transgenic zebrafish (Danio rerio) line (Tg(vtg1:mCherry)) suitable for the direct detection of estrogenicity in environmental samples

Environmental estrogens are a serious concern worldwide due to their ubiquity and adverse ecotoxicological and health effects. Chemical structure of these substances is highly diverse, therefore estrogenicity cannot be predicted on the basis of molecular structure. Furthermore, estimation of estrogenicity of environmental samples based on chemical analytics of suspects is difficult given the complex interaction of chemicals and the impact on estrogenicity. The full estrogenic impact of an environmental sample can thus only be revealed by a series of sensitive in vitro and in vivo ecotoxicological tests. Herein we describe a vitellogenin reporter transgenic zebrafish line (Tg(vtg1:mCherry)) that enables the detection of estrogenicity in the environmentally relevant, low concentration ranges in embryonic tests that are in accordance with 3Rs and relevant animal welfare regulations.The transgene construct used for the development of Tg(vtg1:mCherry) carried a long (3.4 kbp) natural vitellogenin-1 promoter sequence with a high number of ERE sites.A test protocol was developed based on our finding that the endogenous vitellogenin and the reporter show similar spatial expression pattern and both endogenous and vitellogenin reporter is only produced in the left hepatic lobe of 5 dpf zebrafish embryos.Seven generations of Tg(vtg1:mCherry) have been established, and the estrogen responsiveness was tested with different estrogenic substances and wastewater samples. Embryos were exposed from 3 to 5 days post fertilization (dpf). Fluorescence in embryos could be detected upon treatment with 17-ß-estradiol from a concentration of 100 ng/L, 17-α-ethynilestradiol from 1 ng/L, zearalenone from 100 ng/L and bisphenol-A from 1 mg/L. In the adult stage transgene activity appeared to be more sensitive to estrogen treatment, with detectable transgene activity from 5 ng/L 17-ß-estradiol concentration. The transgenic line Tg(vtg1:mCherry) was also suitable for the direct measurement of estrogenicity in wastewater samples without sample extraction. The detection of estrogenic activity using the reporter line was confirmed by the bioluminescent yeast estrogen screen.

High doses of CRISPR/Cas9 ribonucleoprotein efficiently induce gene knockout with low mosaicism in the hydrozoan Clytia hemisphaerica through microhomology-mediated deletion

Targeted mutagenesis using CRISPR/Cas9 technology has been shown to be a powerful approach to examine gene function in diverse metazoan species. One common drawback is that mixed genotypes, and thus variable phenotypes, arise in the F0 generation because incorrect DNA repair produces different mutations amongst cells of the developing embryo. We report here an effective method for gene knockout (KO) in the hydrozoan Clytia hemisphaerica, by injection into the egg of Cas9/sgRNA ribonucleoprotein complex (RNP). Expected phenotypes were observed in the F0 generation when targeting endogenous GFP genes, which abolished fluorescence in embryos, or CheRfx123 (that codes for a conserved master transcriptional regulator for ciliogenesis) which caused sperm motility defects. When high concentrations of Cas9 RNP were used, the mutations in target genes at F0 polyp or jellyfish stages were not random but consisted predominantly of one or two specific deletions between pairs of short microhomologies flanking the cleavage site. Such microhomology-mediated (MM) deletion is most likely caused by microhomology-mediated end-joining (MMEJ), which may be favoured in early stage embryos. This finding makes it very easy to isolate uniform, largely non-mosaic mutants with predictable genotypes in the F0 generation in Clytia, allowing rapid and reliable phenotype assessment.

New fluorescent protein reporters for use with the Drosophila Gal4 expression system and for vital detection of balancer chromosomes.

Since its introduction as a vital marker for gene expression in the mid-1990s (Chalfie et al., 1994), the Aequorea victoria green fluorescent protein (GFP) and its derivatives have become an invaluable research tool to investigators working in a multitude of experimental systems. The molecule’s small size (238 amino acids) and stability enable it to function when fused to a wide variety of proteins and protein localization domains, allowing for nuclear, membrane-bound, cytoskeletal, and organelle-specific vital imaging at a number of wavelengths and with emission in several visually separable spectra (reviewed by De Giorgi et al., 1999). The potential for fluorescence resonance energy transfer (FRET) between different GFP variants, such as the cyan (CFP) and yellow (YFP) fluorescent proteins, additionally allows the use of these markers to detect protein–protein interactions within living cells (Pollok and Heim, 1999). One of the fluorescent proteins’ most useful functions, however, remains that of a simple vital reporter for gene expression and a marker for discrete cell types within the living organism. Although the fluorescent proteins have been used with good success in Drosophila, their performance has sometimes been disappointing when used in attempts to visualize gene expression in the developing Drosophila embryo (e.g., Davis et al., 1995). This is largely attributable to two factors: the time-lag between expression of GFP and its acquisition of fluorescence (Heim et al., 1994), and the autofluorescence of the yolk. The latter problem can be partially overcome by judicious selection of barrier filters or by confocal microscopy, while the former has been mitigated to a degree by the development of “enhanced” fluorescent protein derivatives that are both brighter and quicker to acquire fluorescence than the wild-type GFP (Cormack et al., 1996; Heim et al., 1994, 1995; Heim and Tsien, 1996). Nevertheless, detection of fluorescence can still be difficult in the rapidly developing embryo. We describe here a set of dicistronic GFP and YFP reporters constructed for use with the GAL4/UAS expression system (Brand and Perrimon, 1993), which, as a result of their containing two copies of the respective fluorescent protein gene, are significantly brighter and thus more easily and rapidly detectable than currently available UAS-GFP fly stocks (Dickson, 1996; Yeh et al., 1995). We also describe a set of GFP-marked balancer chromosomes with robust embryonic expression that is detectable at an earlier timepoint than existing GFP balancers (Casso et al., 2000). Transgenic flies were created in which two copies of the “enhanced” fluorescent protein derivatives EGFP or EYFP were separated by the internal ribosome entry site (IRES) from the Drosophila Ubx gene (Hart and Bienz, 1996) and regulated by means of the GAL4/UAS expression system (Fig. 1). Lines on each chromosome that exhibited bright fluorescence when crossed to Gal4containing flies were chosen as the UAS-2xEGFP and UAS-2xEYFP stocks (Table 1) and tested in a variety of settings by examining fluorescence in embryos, egg chambers, larvae, and imaginal discs (Fig. 2a–h, and data not shown). Fluorescence intensity was significantly brighter than that seen when the identical Gal4 drivers were crossed to flies containing the UAS-GFP[S65T] reporter construct (Dickson, 1996; data not shown). This increase in intensity is partially due to the brighter nature of the EGFP derivative compared to the S65T derivative (Cormack et al., 1996) and partially due to the presence of the extra copy of EGFP in the dicistronic construct. Flow cytometric analysis confirms that the 2xEGFP construct is roughly two-fold brighter than a single-copy EGFP construct when expressed in S2 cells (Halfon, 1998). The UAS-2xEYFP flies should be useful in conjunction with CFP for FRET-based experiments and should also be particularly well-suited for imaging embryonic structures that lie near to the autofluorescent gut, as EYFP can be detected under conditions where autofluorescence is minimal (e.g., Leica YFP filter set,

Dynamic visualization of nervous system in live Drosophila.

We have constructed transgenic Drosophila melanogaster lines that express green fluorescent protein (GFP) exclusively in the nervous system. Expression is controlled with transcriptional regulatory elements present in the 5' flanking DNA of the Drosophila Na(+), K(+)-ATPase beta-subunit gene Nervana2 (Nrv2). This regulatory DNA is fused to the yeast transcriptional activator GAL4, which binds specifically to a sequence motif termed the UAS (upstream activating sequence). Drosophila lines carrying Nrv2-GAL4 transgenes have been genetically recombined with UAS-GFP (S65T) transgenes (Nrv2-GAL4+UAS-GFP) inserted on the same chromosomes. We observe strong nervous system-specific fluorescence in embryos, larvae, pupae, and adults. The GFP fluorescence is sufficiently bright to allow dynamic imaging of the nervous system at all of these developmental stages directly through the cuticle of live Drosophila. These lines provide an unprecedented view of the nervous system in living animals and will be valuable tools for investigating a number of developmental, physiological, and genetic neurobiological problems.

Nondestructive indicator of ethoxyresorufin‐O‐deethylase activity in embryonic fish

AbstractMany environmental contaminants of concern produce aryl hydrocarbon (Ah) receptor‐mediated effects often assessed as ethoxyresorufin‐O‐deethylase (EROD) activity. We modified a standardized embryonic and larval fish bioassay using an estuarine fish species (Fundulus heteroclitus) to include measurement of fluorescence in the urinary bladders of living embryos as a nondestructive indicator of EROD activity. This fluorescence was observed only in embryos exposed to the EROD substrate and Ah receptor agonists and was consistent in fluorescence characteristics with the EROD product, resorufin. Aryl hydrocarbon receptor‐agonist exposures at sublethal concentrations in the parts per billion range, produced significant increases in EROD fluorescence in embryos as young as 3 d postfertilization. These findings are consistent with the conclusion that this fluorescence method is a specific and sensitive nondestructive indicator of Ah receptor‐mediated effects in an embryonic fish. As such, this simple method may be useful for assessing effects of environmental contaminants on the early development of fishes and to link molecular indicators with organism‐level responses.

Otoconial formation in the chick: changing patterns of tetracycline incorporation during embryonic development and after hatching

The antibiotic tetracycline (TC) is incorporated into calcifying tissues and serves as a fluorescent marker for identifying calcifying sites in bone and otoconia. Fluorescent labeling was performed at different stages in chick embryos and newly hatched chicks. The stagewise changes in the intensity, location and time course of fluorescent labeling were assessed. TC/egg (1–2 mg) was injected into the yolk sacs of embryos on the 4th, 8th and 11th embryonic days (ED), and the embryos were then killed at specified times after injection. In newly hatched chicks, TC was injected daily intraperitoneally with 0.06 mg TC/g body weight for 7 days and the chicks were killed on the 8th day after hatching. Embryos injected on the 4th ED and killed on the 6th ED showed intense fluorescence in the saccular otoconial layer. All maculae from embryos killed after injection on the 4th ED showed uniformly intense fluorescence throughout the otoconial layer. By the 9th ED, otoconia in all three maculae (saccular, utricular, and lagenar) fluoresced. Maculae from embryos killed after injection on the 8th ED showed uniformly intense fluorescence throughout the otoconial layer or intense localized fluorescence mainly in the upper half of the otoconial layer. All maculae from embryos killed after injection on the 11th ED showed moderate to weak fluorescence primarily in the lower half of the otoconial layer. All maculae from posthatched chicks showed very weak fluorescence throughout the otoconial layer. Otoconia at the periphery of the maculae generally showed weak fluorescence in embryos that had been injected on the 4th and 8th ED, but not in embryos injected on the 11th ED. TC, which competes for calcium binding sites, may inhibit the formation of some otoconia. The formation of giant otoconia may reflect subtle changes in the crystallization microenvironment on these occasions. In brief, the results suggest that: (a) otoconial formation in the saccule precedes that of the utricle and lagena; (b) otoconial formation occurs during the early period (beginning the 6th ED); (c) otoconial formation is stratified, with those in the upper layer forming first and those in the lower layer forming last.