Gene activation via Cre/lox-mediated excision in cowpea (Vigna unguiculata)

Key message Expression of Cre recombinase by AtRps5apro or AtDD45pro enabled Cre/lox-mediated recombination at an early embryonic developmental stage upon crossing, activating transgenes in the hybrid cowpea and tobacco. Abstract Genetic engineering ideally results in precise spatiotemporal control of transgene expression. To activate transgenes exclusively in a hybrid upon fertilization, we evaluated a Cre/lox-mediated gene activation system with the Cre recombinase expressed by either AtRps5a or AtDD45 promoters that showed activity in egg cells and young embryos. In crosses between Cre recombinase lines and transgenic lines harboring a lox-excision reporter cassette with ZsGreen driven by the AtUbq3 promoter after Cre/lox-mediated recombination, we observed complete excision of the lox-flanked intervening DNA sequence between the AtUbq3pro and the ZsGreen coding sequence in F1 progeny upon genotyping but no ZsGreen expression in F1 seeds or seedlings. The incapability to observe ZsGreen fluorescence was attributed to the activity of the AtUbq3pro. Strong ZsGreen expression in F1 seeds was observed after recombination when ZsGreen was driven by the AtUbq10 promoter. Using the AtDD45pro to express Cre resulted in more variation in recombination frequencies between transgenic lines and crosses. Regardless of the promoter used to regulate Cre, mosaic F1 progeny were rare, suggesting gene activation at an early embryo-developmental stage. Observation of ZsGreen-expressing tobacco embryos at the globular stage from crosses with the AtRps5aproCre lines pollinated by the AtUbq3prolox line supported the early activation mode.

Advantages of Screening for Glucose Tolerance in the Sequential Weeks of Gestation

Prelife exposure relates to development during the time preceding the first appearance of life, a time course from “conception to confinement.” From single cell zygote to finally formed fetus at confinement, a remarkable change occurs due to maternal fuels and hormonal influence on the fetal development. The crucial period in the fetal development is the first trimester. Early exposure to aberrant maternal metabolism in the embryonic developmental stage would result in congenital malformation and fetal wastage. Maintaining maternal glucose at the recommended level of fasting 80 to 90 mg and 2 hours postprandial plasma glucose 110 to 120 mg/dL during preconceptional period and throughout pregnancy is the assurance for the healthy offspring with ideal birth weight of 2.5 to 3.5 kg and prevention of noncommunicable diseases in the future.

Expression of Cholinergic Markers and Characterization of Splice Variants during Ontogenesis of Rat Dorsal Root Ganglia Neurons

Dorsal root ganglia (DRG) neurons synthesize acetylcholine (ACh), in addition to their peptidergic nature. They also release ACh and are cholinoceptive, as they express cholinergic receptors. During gangliogenesis, ACh plays an important role in neuronal differentiation, modulating neuritic outgrowth and neurospecific gene expression. Starting from these data, we studied the expression of choline acetyltransferase (ChAT) and vesicular ACh transporter (VAChT) expression in rat DRG neurons. ChAT and VAChT genes are arranged in a “cholinergic locus”, and several splice variants have been described. Using selective primers, we characterized splice variants of these cholinergic markers, demonstrating that rat DRGs express R1, R2, M, and N variants for ChAT and V1, V2, R1, and R2 splice variants for VAChT. Moreover, by RT-PCR analysis, we observed a progressive decrease in ChAT and VAChT transcripts from the late embryonic developmental stage (E18) to postnatal P2 and P15 and in the adult DRG. Interestingly, Western blot analyses and activity assays demonstrated that ChAT levels significantly increased during DRG ontogenesis. The modulated expression of different ChAT and VAChT splice variants during development suggests a possible differential regulation of cholinergic marker expression in sensory neurons and confirms multiple roles for ACh in DRG neurons, both in the embryo stage and postnatally.

On the Biomechanics of Cardiac S-Looping in the Chick: Insights From Modeling and Perturbation Studies

Cardiac looping is an important embryonic developmental stage where the primitive heart tube (HT) twists into a configuration that more closely resembles the mature heart. Improper looping leads to congenital defects. Using the chick embryo as the experimental model, we study cardiac s-looping wherein the primitive ventricle, which lay superior to the atrium, now assumes its definitive position inferior to it. This process results in a heart loop that is no longer planar with the inflow and outflow tracts now lying in adjacent planes. We investigate the biomechanics of s-looping and use modeling to understand the nonlinear and time-variant morphogenetic shape changes. We developed physical and finite element models and validated the models using perturbation studies. The results from experiments and models show how force actuators such as bending of the embryonic dorsal wall (cervical flexure), rotation around the body axis (embryo torsion), and HT growth interact to produce the heart loop. Using model-based and experimental data, we present an improved hypothesis for early cardiac s-looping.

Estimating cod egg developmental stage based on DNA concentration

Pelagic egg surveys are commonly used to map Atlantic cod spawning areas in the northeastern Atlantic. However, a sampling location may not necessarily indicate a spawning location, because more developed eggs may have drifted long distances in coastal currents. Newly spawned eggs have only a few embryonic cells, whereas eggs in later developmental stages have progressively larger numbers of cells and hence greater amounts of DNA. The progression through developmental stages largely depends on temperature, which influences cell division and growth. Preservation of the eggs for later analysis presents logistical dilemmas. Preservation in ethanol is suitable for DNA extraction but obscures developmental stage, as the eggs shrink and turn opaque. On the other hand, preservation in formaldehyde retains visual characteristics, but this method limits the ability to extract intact DNA. Here we report a method to estimate developmental stages in Atlantic cod eggs by assaying the amount of DNA from eggs preserved in ethanol. We used a correlation between the amount of DNA in an egg and its embryonic developmental stage in samples from western Norway and used this correlation to estimate developmental stages of eggs sampled in northern Norway.

Artificial stripping and embryonic development of the common gudgeon (Gobio gobio L.) and its use in embryo-larval tests – a pilot study

Artificial reproduction with and without hormonal treatment was performed in females of the common gudgeon (Gobio gobio L.) to assess the suitability of this cyprinid species as a fish model for embryo-larval cytotoxic tests. Considering sperm immobilisation tests, the solution for tench was utilizable. The clearing solution designed for the observation of embryonic development in the European catfish was also found suitable for the common gudgeon embryos. The egg and embryonic mortality, embryonic development, hatching and survival rate were examined. Ovulation occurred in 89% of females after the application of the carp pituitary (5 mg/kg) but embryonic mortality reached nearly 100%. The highest embryonic mortality in females with hormonal treatment was observed within 24 hours of development. On the other hand, the reproduction of females without the application of hormonal stimulation was nearly completely successful with mean mortality 60%. The hatching of embryos of females without hormonal stimulation occurred (average values) 71 hours after fertilization and lasted 65 hours. Embryonic development finished within 176 hours. Our pilot study demonstrated that the eggs of common gudgeon were suitable for embryo-larval tests from the 7^th to the 8^th embryonic developmental stage.