Introduction of loxP sites by electroporation in the mouse genome; a simple approach for conditional allele generation in complex targeting loci.

Background: The discovery of the CRISPR-Cas9 system and its applicability in mammalian embryos has revolutionized the way we generate genetically engineered animal models. To date, models harbouring conditional alleles (i.e.: two loxP sites flanking an exon or a critical DNA sequence of interest) remain the most challenging to generate as they require simultaneous cleavage of the genome using two guides in order to properly integrate the repair template. In the current manuscript, we describe a modification of the sequential electroporation procedure described by Horii et al (2017). We demonstrate production of conditional allele mouse models for eight different genes via one of two alternative strategies: either by consecutive sequential electroporation (strategy A) or non-consecutive sequential electroporation (strategy B). Results: By using strategy A, we demonstrated successful generation of conditional allele models for three different genes (Icam1, Lox, and Sar1b), with targeting efficiencies varying between 5 to 13%. By using strategy B, we generated five conditional allele models (Loxl1, Pard6a, Pard6g, Clcf1, and Mapkapk5), with targeting efficiencies varying between 3 to 25%. Conclusion: Our modified electroporation-based approach, involving one of the two alternative strategies, allowed the production of conditional allele models for eight different genes via two different possible paths. This reproducible method will serve as another reliable approach in addition to other well-established methodologies in the literature for conditional allele mouse model generation.

In Vivo Imaging of Mammalian Embryos By NIR-I Photoacoustic Tomography and NIR-II Optical Coherence Tomography Using Gold Nanostars as Multifunctional Contrast Agents

Background High resolution, strong contrast and multimodality visualization of live mammalian embryo is an important requirement for studying foetal development. Photoacoustic Tomography (PAT) and Optical Coherence Tomography (OCT) are two advanced imaging modalities that has been utilized for embryonic imaging. However, high contrast, multiscale and deep tissue visualization of live embryos remains challenging. Results Here, we demonstrate the use of gold nanostars (GNS) as multimodality contrast agents for the visualization and differentiation of embryos in vivo using NIR-I PAT and NIR-II OCT. We perform NIR-I PAT imaging to confirm in vivo GNS accumulation in the foetuses, and then use a customized NIR-II OCT system to further reveal deep, contrast-enhanced micro features of freshly harvested embryos. We investigate two different GNS administration pathways, i.e. intravenous and intravaginal injection, and significant enhancement of signal, image contrast, and imaging depth are achieved for both PAT and OCT. Conclusions These findings prove that PAT-OCT bi-modal imaging with GNS enhancement provides more accurate structural characteristic of live mammalian embryos, and thus reveal its potential for embryonic development visualization and early abnormality examination. These findings prove that PAT-OCT bi-modal imaging with GNS enhancement provides more accurate structural characteristic of live mammalian embryos, and thus reveal its potential for embryonic development visualization and early abnormality examination.

Generation of mutant pigs by lipofection-mediated genome editing in embryos

The specificity and efficiency of CRISPR/Cas9 gene-editing systems are determined by several factors, including the mode of delivery, when applied to mammalian embryos. Given the limited time window for delivery, faster and more reliable methods to introduce Cas9-gRNA ribonucleoprotein complexes (RNPs) into target embryos are needed. In pigs, somatic cell nuclear transfer using gene-modified somatic cells and the direct introduction of gene editors into the cytoplasm of zygotes/embryos by microinjection or electroporation have been used to generate gene-edited embryos; however, these strategies require expensive equipment and sophisticated techniques. In this study, we developed a novel lipofection-mediated RNP transfection technique that does not require specialized equipment for the generation of gene-edited pigs and produced no detectable off-target events. In particular, we determined the concentration of lipofection reagent for efficient RNP delivery into embryos and successfully generated MSTN gene-edited pigs (with mutations in 7 of 9 piglets) after blastocyst transfer to a recipient gilt. This newly established lipofection-based technique is still in its early stages and requires improvements, particularly in terms of editing efficiency. Nonetheless, this practical method for rapid and large-scale lipofection-mediated gene editing in pigs has important agricultural and biomedical applications.

The Long Terminal Repeats of ERV6 Are Activated in Pre-Implantation Embryos of Cynomolgus Monkey

Precise gene regulation is critical during embryo development. Long terminal repeat elements (LTRs) of endogenous retroviruses (ERVs) are dynamically expressed in blastocysts of mammalian embryos. However, the expression pattern of LTRs in monkey blastocyst is still unknown. By single-cell RNA-sequencing (seq) data of cynomolgus monkeys, we found that LTRs of several ERV families, including MacERV6, MacERV3, MacERV2, MacERVK1, and MacERVK2, were highly expressed in pre-implantation embryo cells including epiblast (EPI), trophectoderm (TrB), and primitive endoderm (PrE), but were depleted in post-implantation. We knocked down MacERV6-LTR1a in cynomolgus monkeys with a short hairpin RNA (shRNA) strategy to examine the potential function of MacERV6-LTR1a in the early development of monkey embryos. The silence of MacERV6-LTR1a mainly postpones the differentiation of TrB, EPI, and PrE cells in embryos at day 7 compared to control. Moreover, we confirmed MacERV6-LTR1a could recruit Estrogen Related Receptor Beta (ESRRB), which plays an important role in the maintenance of self-renewal and pluripotency of embryonic and trophoblast stem cells through different signaling pathways including FGF and Wnt signaling pathways. In summary, these results suggest that MacERV6-LTR1a is involved in gene regulation of the pre-implantation embryo of the cynomolgus monkeys.

Δ9-Tetrahydrocannabinol inhibits Hedgehog-dependent patterning during development

ABSTRACT Many developmental disorders are thought to arise from an interaction between genetic and environmental risk factors. The Hedgehog (HH) signaling pathway regulates myriad developmental processes, and pathway inhibition is associated with birth defects, including holoprosencephaly (HPE). Cannabinoids are HH pathway inhibitors, but little is known of their effects on HH-dependent processes in mammalian embryos, and their mechanism of action is unclear. We report that the psychoactive cannabinoid Δ9-tetrahydrocannabinol (THC) induces two hallmark HH loss-of-function phenotypes (HPE and ventral neural tube patterning defects) in Cdon mutant mice, which have a subthreshold deficit in HH signaling. THC therefore acts as a ‘conditional teratogen’, dependent on a complementary but insufficient genetic insult. In vitro findings indicate that THC is a direct inhibitor of the essential HH signal transducer smoothened. The canonical THC receptor, cannabinoid receptor-type 1, is not required for THC to inhibit HH signaling. Cannabis consumption during pregnancy may contribute to a combination of risk factors underlying specific developmental disorders. These findings therefore have significant public health relevance.

1. What are stem cells?

‘What are stem cells?’ explains that a stem cell is a cell that can both reproduce itself and generate offspring of different functional cell types and begins by considering the nature of cells in general, wherein cells are understood to be the ultimate structural unit of an animal or plant body. Stem cells in the body persist long term, usually for the lifetime of the organism. Good examples of differentiated cells arising from stem cells are those of the skin, the blood, and the lining of the intestine. Embryonic stem cells are grown in culture from early mammalian embryos. The reason that stem cell research is seen as the source for new cures is largely because this technology offers a route to cell therapy.

Impact of Global Transcriptional Silencing on Cell Cycle Regulation and Chromosome Segregation in Early Mammalian Embryos

The onset of an early development is, in mammals, characterized by profound changes of multiple aspects of cellular morphology and behavior. These are including, but not limited to, fertilization and the merging of parental genomes with a subsequent transition from the meiotic into the mitotic cycle, followed by global changes of chromatin epigenetic modifications, a gradual decrease in cell size and the initiation of gene expression from the newly formed embryonic genome. Some of these important, and sometimes also dramatic, changes are executed within the period during which the gene transcription is globally silenced or not progressed, and the regulation of most cellular activities, including those mentioned above, relies on controlled translation. It is known that the blastomeres within an early embryo are prone to chromosome segregation errors, which might, when affecting a significant proportion of a cell within the embryo, compromise its further development. In this review, we discuss how the absence of transcription affects the transition from the oocyte to the embryo and what impact global transcriptional silencing might have on the basic cell cycle and chromosome segregation controlling mechanisms.

A robust ex vivo system to study cellular dynamics underlying mouse peri-implantation development

Upon implantation, mammalian embryos undergo major morphogenesis and key developmental processes such as body axis specification and gastrulation. However, limited accessibility obscures study of these crucial processes. Here, we develop an ex vivo Matrigel-collagen-based culture to recapitulate mouse development from E4.5 to 6.0. Our system not only recapitulates embryonic growth, axis initiation, and overall 3D architecture in 49% of cases, its compatibility with light-sheet microscopy enables study of cellular dynamics through automatic cell segmentation. We find that upon implantation, release of the increasing tension in the polar trophectoderm is necessary for its constriction and invagination. The resulting extra-embryonic ectoderm plays a key role in growth, morphogenesis and patterning of the neighboring epiblast, which subsequently gives rise to all embryonic tissues. This 3D-ex vivo system thus offers an unprecedented access to peri-implantation development for in toto monitoring, measurement and spatio-temporally controlled perturbation, revealing a mechano-chemical interplay between extra-embryonic and embryonic tissues.