Application of Transposon Systems in The Transgenesis of Bovine Somatic And Germ Cells

Background: Several DNA transposons, PiggyBac (PB), Sleeping beauty (SB) and Tol2 have been applied as effective means for transgenesis in many species. Cattle are not typical experimental animals, and relatively little verification has been studied in this species. Thus, the goal of this study was the applicability of three transposon systems in somatic and embryo cells in cattle, while also determining which of the three systems is appropriate for each type of cell. To conduct the experiment, green fluorescent protein (GFP)-expressing transposon systems were used for electroporation and microinjection in the somatic cells and embryo stage, respectively. After transfection, GFP-positive cells or blastocysts were observed through a fluorescent microscope and transfection efficiency was calculated by FACS.Results: In the bovine somatic cells experiment, the PB (63.97 ± 11.56) showed higher efficiency as compared to the other two systems (SB: 50.74 ± 13.02 and Tol2: 16.55 ± 5.96). Unlike the results of the somatic cells, Tol2 (75.00%) and SB (70.00%) in the embryo were more efficient as compared to PB (42.86 %).Conclusions: These results demonstrate that all three transposon systems can be used in bovine somatic cells and embryos as a gene engineering experimental method and which type of transposon system is appropriate to apply depending on the cell type.

Developmental capacity is unevenly distributed among single blastomeres of 2-cell and 4-cell stage mouse embryos

During preimplantation development, mammalian embryo cells (blastomeres) cleave, gradually losing their potencies and differentiating into three primary cell lineages: epiblast (EPI), trophectoderm (TE), and primitive endoderm (PE). The exact moment at which cells begin to vary in their potency for multilineage differentiation still remains unknown. We sought to answer the question of whether single cells isolated from 2- and 4-cell embryos differ in their ability to generate the progenitors and cells of blastocyst lineages. We revealed that twins were often able to develop into blastocysts containing inner cell masses (ICMs) with PE and EPI cells. Despite their capacity to create a blastocyst, the twins differed in their ability to produce EPI, PE, and TE cell lineages. In contrast, quadruplets rarely formed normal blastocysts, but instead developed into blastocysts with ICMs composed of only one cell lineage or completely devoid of an ICM altogether. We also showed that quadruplets have unequal capacities to differentiate into TE, PE, and EPI lineages. These findings could explain the difficulty of creating monozygotic twins and quadruplets from 2- and 4-cell stage mouse embryos.

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.

Select Phytochemicals Reduce Campylobacter jejuni in Postharvest Poultry and Modulate the Virulence Attributes of C. jejuni

Consumption or handling of poultry and poultry products contaminated with Campylobacter species are a leading cause of foodborne illness in humans. Current strategies employed to reduce Campylobacter in live chickens provide inconsistent results indicating the need for an alternative approach. This study investigated the efficacy of phytochemicals, namely, turmeric, curcumin, allyl sulfide, garlic oil, and ginger oil, to reduce Campylobacter jejuni in postharvest poultry and sought to delineate the underlying mechanisms of action. Two experiments were conducted on the thigh skin of the chicken, and each experiment was repeated twice. Samples were inoculated with 50 μl (∼107 CFU/sample) of C. jejuni strain S-8 and allowed to adhere for 30 min. Skin samples were dipped into their respective prechilled treatment solutions (0.25 and 0.5% in experiments 1 and 2, respectively) at 4°C for an hour to simulate chilling tank treatment, followed by plating to enumerate C. jejuni (n = 3 samples/treatment/trial). The mechanisms of action(s) were investigated using subinhibitory concentration (SIC) in adhesion, quorum sensing, and gene expression analyses. Adhesion assay was conducted on the monolayers of ATCC CRL-1590 chicken embryo cells challenged with C. jejuni and incubated in the presence or absence of phytochemicals for 1.5 h, followed by plating to enumerate adhered C. jejuni. The effects of phytochemicals on quorum sensing and cell viability were investigated using Vibrio harveyi bioluminescence and LIVE/Dead BacLightTM bacterial viability assays, respectively. In addition, droplet digital PCR determined the gene expression analyses of C. jejuni exposed to phytochemicals. Data were analyzed by GraphPad Prism version 9. C. jejuni counts were reduced by 1.0–1.5 Log CFU/sample with garlic oil or ginger oil at 0.25 and 0.5% (p < 0.05). The selected phytochemicals (except curcumin) reduced the adhesion of C. jejuni to chicken embryo cells (p < 0.05). In addition, all the phytochemicals at SIC reduced quorum sensing of C. jejuni (p < 0.05). The cell viability test revealed that cells treated with 0.25% of phytochemicals had compromised cell membranes indicating this as a mechanism that phytochemicals use to damage/kill C. jejuni. This study supports that the application of phytochemicals in postharvest poultry would significantly reduce C. jejuni in poultry meat.

In Vitro Study of the Effect of HBV Infection on Early Embryonic Development

Objective: This study aimed to clarify the infection state of early embryos by HBV and the effect of HBV infection on embryo development. Methods: In vitro culture was carried out on the fresh surplus embryos from infertile couples on the third day (D3) who received routine in vitro fertilization and embryo transfer (IVF-ET) treatment. The embryo cells were infected in vitro by the serum of HBV infected patients with different viral loads. HBsAg quantity in the supernatant secreted by embryo and HBV mRNA in embryo cells were detected. The development of embryo cells after HBV infection was evaluated by embryo morphological parameter score and fragment score in embryo cells. Results: From May 2013 to July 2015, 238 cases of D3 fresh surplus embryos from 120 infertile couples and 128 samples of embryo culture supernatant were collected. The HBsAg positive rate in the supernatant of embryos in the high viral load infection group was significantly higher than that in the low viral load infection group (40.0% vs.15.6%, p<0.05). HBV mRNA was detected in 2 of 17 HBsAg positive embryos. The scores of embryos in the high load HBV culture solution group were higher than those in the low load group (p=0.04). There was one formed blastocyst in low load HBV culture group, but there was no blastocyst formation in all embryo cells of high load group (p<0.001).Conclusion: The results suggest that embryo cells can be directly infected by HBV in vitro. HBV infection affects the development of embryos.

Frequent loss of heterozygosity in CRISPR-Cas9–edited early human embryos

CRISPR-Cas9 genome editing is a promising technique for clinical applications, such as the correction of disease-associated alleles in somatic cells. The use of this approach has also been discussed in the context of heritable editing of the human germ line. However, studies assessing gene correction in early human embryos report low efficiency of mutation repair, high rates of mosaicism, and the possibility of unintended editing outcomes that may have pathologic consequences. We developed computational pipelines to assess single-cell genomics and transcriptomics datasets from OCT4 (POU5F1) CRISPR-Cas9–targeted and control human preimplantation embryos. This allowed us to evaluate on-target mutations that would be missed by more conventional genotyping techniques. We observed loss of heterozygosity in edited cells that spanned regions beyond the POU5F1 on-target locus, as well as segmental loss and gain of chromosome 6, on which the POU5F1 gene is located. Unintended genome editing outcomes were present in ∼16% of the human embryo cells analyzed and spanned 4–20 kb. Our observations are consistent with recent findings indicating complexity at on-target sites following CRISPR-Cas9 genome editing. Our work underscores the importance of further basic research to assess the safety of genome editing techniques in human embryos, which will inform debates about the potential clinical use of this technology.

Migration of Oil Bodies in Embryo Cells during Acquisition of Desiccation Tolerance in Chemically Defoliated Corn (Zea mays L.) Seed Production Fields

Chemical defoliation of seed corn production fields accelerates seed maturation and desiccation and expedites seed harvest. Early seed harvest is important to minimize the risk of frost damage while in the field. This newly adopted seed production practice also allows seed companies to plan harvest and manage dryer space more efficiently. However, premature defoliation may interfere with the migration of oil bodies within embryo cells during desiccation and affect seed germination and vigor. The objective of this study was to investigate the effect of chemical defoliation on the migration patterns of oil bodies within embryo cells during desiccation. Chemically defoliated and non-defoliated plants from five commercial hybrid seed corn fields were sampled in 2014 and 2015. Whole ears with husks were harvested before and after defoliant application at 600 g H2O kg−1 fresh weight (fw), and weekly thereafter until seed reached approximately 300–350 g H2O kg−1 fw. Ten embryos extracted from center-row seeds were fixed to stop metabolic processes, then sliced, processed, and photographed using scanning transmission electron microscopy. The oil bodies within embryo cells followed normal migration patterns according to seed moisture content, regardless of defoliation treatment. Seed germination and vigor were verified and were not significantly affected by defoliation. Chemical defoliation is a viable production practice to accelerate seed corn desiccation and to manage harvest and seed dryer availability more efficiently without negatively affecting seed germination and vigor.

Molecular Characterization of Paralichthys olivaceus MAF1 and Its Potential Role as an Anti-Viral Hemorrhagic Septicaemia Virus Factor in Hirame Natural Embryo Cells

MAF1 is a global suppressor of RNA polymerase III-dependent transcription, and is conserved from yeast to human. Growing evidence supports the involvement of MAF1 in the immune response of mammals, but its biological functions in fish are unknown. We isolated and characterized Maf1 from the olive flounder Paralichthys olivaceus (PoMaf1). The coding region of PoMaf1 comprised 738 bp encoding a 245-amino-acid protein. The deduced PoMAF1 amino acid sequence shared features with those of MAF1 orthologues from vertebrates. PoMaf1 mRNA was detected in all tissues examined, and the levels were highest in eye and muscle tissue. The PoMaf1 mRNA level increased during early development. In addition, the PoMaf1 transcript level decreased during viral hemorrhagic septicemia virus (VHSV) infection of flounder hirame natural embryo (HINAE) cells. To investigate the role of PoMaf1 in VHSV infection, single-cell-derived PoMaf1 knockout HINAE cells were generated using the clustered regularly interspaced short palindromic repeats/CRISPR-associated-9 (CRISPR/Cas9) system, and cell clones with complete disruption of PoMaf1 were selected. PoMaf1 disruption increased the VHSV glycoprotein (G) mRNA levels during VHSV infection of HINAE cells, implicating PoMAF1 in the immune response to VSHV infection. To our knowledge, this is the first study to characterize fish Maf1, which may play a role in the response to viral infection.