Literatura científica selecionada sobre o tema "ADN recombiné – Embryons"

Somatic cells in semen are a potential source of nuclei for nuclear transfer to produce genetically identical animals. This is especially important when an animal has died and the only viable genetic material available is frozen semen. In our previous studies, epithelial cells were cultured from fresh ovine and bovine ejaculates and blastocyst stage embryos were produced using these cells (Liu J et al. 2007 Biol. Reprod. special issue, 177; Liu J et al. 2008 Reprod. Fertil. Dev. 20, 102). However, growing cells from frozen semen can be difficult. We hypothesized that nuclei of the somatic cells in frozen semen can be used for nuclear transfer even though the cell membrane and cytoplasm are damaged during the semen freezing process. Electrical fusion or piezo assisted direct injection was applied to introduce nuclei of somatic cells isolated from frozen bovine semen (not cultured) into enucleated bovine oocytes. With electrical fusion, only 5 of the 64 (7.8%) recombined couplets fused, which is lower than our normal fusion rate of approximately 70%. Of the 5 fused embryos, one cleaved and developed to a 4-cell stage embryo. Staining with propidium iodide indicated that less than 10% of somatic cells isolated from frozen bovine semen were viable. These results suggest that it might not be practical to introduce nuclei of the cells in frozen semen into oocytes by fusion due to the high proportions of dead cells. Membranes of the cells in frozen semen were hard and difficult to break by piezo pulses or drawing in and out of the injection pipette. Therefore, whole cell intracytoplasmic injection was applied. After couplet recombination, activation was induced by applying two 0.3 kV cm–1, 55 μs direct-current pulses delivered by an Eppendorf Multiporator (Eppendorf, North America) in activation medium that was composed of 0.28 m Mannitol (Sigma-Aldrich, St. Louis, MO, USA), 0.1 mm CaCl2 (Sigma-Aldrich), and 0.1 mm MgSO4 (Sigma-Aldrich), followed by incubation in 10 μg mL–1 cycloheximide (Sigma-Aldrich) and 5 μg mL–1 cytochalasin B (Sigma-Aldrich) for 5 h in a humidified 5% CO2, 5% O2, and 90% N2 gas mixture at 38.5°C. Of the 299 recombined embryos, 82 (27.4%) either cleaved or were fragmented. Of the 82 embryos, 48 were examined and 42 (87.5%) were determined to be fragmented (contained intact donor cells). Low cleavage rates were observed in embryos produced by direct injection (4.5%), but no further embryonic development occurred. Cells cultured from fresh bovine semen were used as positive controls for whole cell intracytoplasmic injection. Of the 74 recombined embryos, 73 (98.6%) cleaved and 4 developed into blastocysts. These results highlight the difficulty of obtaining viable embryos by employing nuclear transfer and somatic cells obtained from frozen semen samples. Additional research is warranted given the potential value of this approach for recovering lost genetics.

Abstract BACKGROUND Human gastrointestinal (GI) organoid technologies have transformed GI disease research. By recapitulating the essential steps that occur during embryonic organ development, we could generate in vitro human colonic organoids (HCOs) (Munera et al, Cell Stem Cell. 2017) and human intestinal organoids (HIOs) (Spence et al, Nature. 2011) from iPSCs. Interestingly, HCOs contain both epithelial and surrounding mesenchymal derivatives, including myofibroblasts and tissue-resident immune cells. Our preliminary data demonstrate that HCOs co-develop CD163 positive macrophages derived from the hemogenic endothelium that develops within the colonic mesenchyme. Conversely, HIOs do not spontaneously develop tissue-resident immune cells. OBJECTIVE To incorporate tissue-resident immune cells into HIOs and use this new model to interrogate diseases such as IBD and Crohn’s Disease. RESULTS HIOs did not form endothelial tubes and immune cell floaters as seen in HCOs under the microscope 20 days after the start of differentiation. Moreover, HIO supernatants yielded few to no CFUs in Methocult assays compared to HCOs. Therefore, we tried two approaches to generate a novel HIO model system containing resident macrophages. First, we tried to transfer mesenchymal cells innately present in HCOs. We dissociate mesenchyme from HCOs and recombine them with HIO epithelium around day 20 from the start of differentiation (Figure 1A). We could successfully dissociate HCO-mesenchymal components from their epithelium and recombined them with HIO epithelium, but we could not detect CD163 positive macrophages in recombined HIOs. Second, we tried to transfer the supernatant of HCOs to HIOs around day 20 from the start of differentiation (Figure 1B). We confirmed the presence of CD163+ macrophages in day 35 HIOs using immunostaining and RNA sequencing (Figure 2A, B). We also confirmed the expression of cytokines such as IL10, IL12, and TGFβ via qPCR. Furthermore, we wanted to investigate whether these tissue resident macrophages were maintained after further maturation. As previously described, HIO transplantation into the mouse kidney capsule for 12 weeks allows for tissue maturation enabling crypt/villus formation and smooth muscle differentiation (Figure 2C) (Watson et al, Nat. Med. 2014). After recombined HIOs were transplanted, human CD163+ macrophages displayed expansion and were not displaced by bone marrow derived mouse macrophages (expressing F4/80). In the future, we also plan to differentiate monocytes directly from iPSCs and inject the monocytes into HIOs to see if they resident in the tissue as macrophages (Figure 1C). CONCLUSION We generated HIOs containing tissue-resident macrophages by transferring supernatant of HCOs. We expect to accelerate our understanding of the mechanisms of IBD such as CD because of the development of HIOs technology containing of immune cells.

AbstractThe developmental functions of oocytes of three strains of mice (Kunming, ICR and C57BL/6-Tg(CAG-EGFP)C14-Y01-FM131Osb) recombined with the nuclei of first polar bodies (Pbs I) were explored. Cumulus oocyte complexes (COCs) from the mice were collected after superovulation, then Pbs I were obtained from the COCs by 2% pronase treatment. The survival of Pbs I under different temperatures was identified by morphology and trypan blue staining. Later, the polar body I (Pb 1) nucleus and a little cytoplasm was injected into each oocyte, the nuclei of which had been enucleated by micromanipulation. Oocytes recombined with Pbs I were fertilized, then cultured in vitro in order to observe their further development. The results showed that the vigour of Pbs I was maintained for 12–14 h after superovulation, and was still maintained after 48 h at 4 °C. A total of 13 out of 117 recombined oocytes from Kunming and ICR mice, as well as 3 out of 38 recombined oocytes from C57BL/6-Tg(CAG-EGFP)C14-Y01-FM131Osb mice, developed into two-cell embryos. The experiments confirmed that mouse oocytes recombined with the nuclei of Pbs I could maintain fertilization and development. These results present valuable references for further utilization of genetic resources for farm animals

Abstract The activities of transcriptional complexes drive the proper development and function of insulin producing beta-cells, ultimately required for the regulation of glucose homeostasis. Our prior work helped to establish that the LIM-homeodomain transcription factor (TF), Islet-1 (Isl1), directly interacts with the Ldb1 co-regulator in developing and adult beta-cells. We further found that a member of the Single Stranded DNA-Binding Protein (SSBP) co-regulator family, SSBP3, interacts with the Isl1:Ldb1 complex in beta-cells and primary islets to impact critical target genes MafA and Glp1R. Members of the SSBP family of co-regulators stabilize TF complexes in various tissues, ranging from brain to skin, by binding directly to Ldb1 and protecting the factors from ubiquitin-mediated turnover. Because of this, we hypothesized that SSBP3 would have similarly critical roles as Isl1 and Ldb1 for beta-cell development and function in vivo. To assess this, we first developed a novel SSBP3 floxed mouse line, where Cre-mediated recombination is predicted to impart loss of the Ldb1-interacting domain, plus an early termination. We bred this mouse into a Pax6-Cre transgenic line to recombine SSBP3 in the developing pancreatic islet, a model termed SSBP3islet. We found that SSBP3islet neonates become progressively hyperglycemic and both male and female mice are glucose intolerant as early as postnatal day (P) 21. These results are similar to previous Ldb1 and Isl1 knockouts in the embryonic islet, both of which were hyperglycemic by P10. We observed a reduction of the beta-cell maturity marker, MafA, and disruptions in islet cell architecture with an apparent increase in both glucagon+ alpha-cells and ghrelin+epsilon-cells at P10 and P28. In ongoing studies we are generating embryonic day (E)18.5 embryos to determine islet development defects and will conduct chromatin immunoprecipitation (ChIP) experiments to determine the beta-cell and islet genes directly bound by SSBP3 in vivo. These experiments will further elucidate the regulation of islet function by LIM complexes, knowledge that is central not only for our understanding of glucose homeostasis but for the development of novel diabetes therapeutics.

Abstract Cytoplasmic incompatibility is a selfish reproductive manipulation induced by the endosymbiont Wolbachia in arthropods. In males Wolbachia modifies sperm, leading to embryonic mortality in crosses with Wolbachia-free females. In females, Wolbachia rescues the cross and allows development to proceed normally. This provides a reproductive advantage to infected females, allowing the maternally transmitted symbiont to spread rapidly through host populations. We identified homologs of the genes underlying this phenotype, cifA and cifB, in 52 of 71 new and published Wolbachia genome sequences. They are strongly associated with cytoplasmic incompatibility. There are up to seven copies of the genes in each genome, and phylogenetic analysis shows that Wolbachia frequently acquires new copies due to pervasive horizontal transfer between strains. In many cases, the genes have subsequently acquired loss-of-function mutations to become pseudogenes. As predicted by theory, this tends to occur first in cifB, whose sole function is to modify sperm, and then in cifA, which is required to rescue the cross in females. Although cif genes recombine, recombination is largely restricted to closely related homologs. This is predicted under a model of coevolution between sperm modification and embryonic rescue, where recombination between distantly related pairs of genes would create a self-incompatible strain. Together, these patterns of gene gain, loss, and recombination support evolutionary models of cytoplasmic incompatibility.

Gynogenesis is a form of parthenogenesis in which eggs require sperm for fertilization but develop to adulthood without the contribution of paternal genome information, which happens naturally in some species. InXenopus, gynogenetic diploid animals can be made experimentally. In mutagenesis strategies that only generate one allele of a recessive mutation, as might occur during gene editing, gynogenesis can be used to quickly reveal a recessive phenotype in eggs carrying a recessive mutation, thereby skipping one generation normally required to screen by conventional genetics.Xenopusoocytes do not complete meiosis until shortly after fertilization, and the second polar body is retained in fertilized eggs. Using ultraviolet (UV)-irradiated sperm, fertilization can be triggered without a genetic paternal contribution. Upon applying cold shock at the proper time to such embryos, ejection of the second polar body can be suppressed and both maternal sister chromatids are retained, leading to the development of gynogenetic diploid embryos. Because the genome of the resultant animals consists of recombined sister chromatids because of crossover events during meiosis, it is not completely homozygous throughout the whole genome. Nevertheless, the genome is homozygous at some loci proximal to the centromere that are unlikely to undergo recombination during meiosis and homozygous at reduced frequency if mutations are farther from the centromere, but still generally at a scorable level. Therefore, this technique is useful for rapid screening phenotypes of recessive mutations in such regions. We describe here a step-by-step protocol to achieve cold shock-mediated gynogenesis inXenopus tropicalis.

The human uterus develops from the distal Mullerian Duct, a derivative of the mesoderm germ layer. Unlike other mammalian species (eg. mouse) the endometrium of the human uterus develops prenatally during gestation. Little is known about the developmental process involved. A better understanding of human endometrial development may shed light on the mechanisms involved in endometrial regeneration and pathogenesis of adult proliferative endometrial diseases. Mouse neonatal uterine mesenchyme (mNUM) is inductive and can maintain the phenotype of normal adult human endometrial epithelial cells [1]. Both adult human endometrial stroma and neonatal mouse endometrial mesenchyme secrete growth factors of the TGF-beta family including BMPs which have been shown to play an important role in differentiation of human embryonic stem cells (HESC) [2, 3]. Hypothesis: mNUM will direct differentiation of HESC to form Mullerian Duct-like epithelium. Aim: to investigate the role of mNUM in differentiating HESC in vitro and in vivo using A tissue recombination technique. Method: Embryoid bodies (EB) were formed from GFP labelled HESC (ENVY) and GFP-MIXL1 HESC reporter line [4, 5] and recombined with 2 × 0.5 mm pieces of day 1 epithelial cell-free mNUM. Recombinant tissues were either harvested for gene expression analysis or grafted under the kidney capsule of NOD/SCID mice. Results: We found by qRT-PCR that mNUM induces HESC to form mesendoderm/mesoderm progenitors in vitro, obligate intermediates of the developing Mullerian Duct. After further incubation in vivo under the guidance of mNUM, HESC differentiated to form duct-like structures comprising mesoepithelial cells that co-expressed several key developmental proteins of the Mullerian Duct including Emx2, Pax2, Hoxa10, CA125, and also intermediate filament markers such as CK8/18, Vimentin (n = 8). Conclusion: Our study demonstrated for the first time that mNUM can direct HESC to form a mesodermally derived epithelium that is Mullerian Duct-like, providing a novel model for studying human uterine development. (1) Kurita T, et al., The activation function-1 domain of estrogen receptor alpha in uterine stromal cells is required for mouse but not human uterine epithelial response to estrogen. Differentiation, 2005. 73(6): 313–22.(2) Hu J, Gray CA, Spencer TE, Gene expression profiling of neonatal mouse uterine development. Biol Reprod, 2004. 70(6): 1870–6.(3) Stoikos CJ, et al., A distinct cohort of the TGFbeta superfamily members expressed in human endometrium regulate decidualization. Hum Reprod, 2008. 23(6): 1447–56.(4) Davis R, et al., Targeting a GFP reporter gene to the MIXL1 locus of human embryonic stem cells identifies human primitive streak-like cells and enables isolation of primitive hematopoietic precursors. Blood, 2008. 111(4): 1876–84.(5) Costa M, et al., The hESC line Envy expresses high levels of GFP in all differentiated progeny. Nat Methods, 2005. 2(4): 259–60.

Abstract The CreERT2/loxP system is widely used to induce conditional gene deletion in mice. One of the main advantages of the system is that Cre-mediated recombination can be controlled in time through Tamoxifen administration. This has allowed researchers to study the function of embryonic lethal genes at later developmental timepoints. In addition, CreERT2 mouse lines are commonly used in combination with reporter genes for lineage tracing and mosaic analysis. In order for these experiments to be reliable, it is crucial that the cell labeling approach only marks the desired cell population and their progeny, as unfaithful expression of reporter genes in other cell types or even unintended labeling of the correct cell population at an undesired time point could lead to wrong conclusions. Here we report that all CreERT2 mouse lines that we have studied exhibit a certain degree of Tamoxifen-independent, basal, Cre activity. Using Ai14 and Ai3, two commonly used fluorescent reporter genes, we show that those basal Cre activity levels are sufficient to label a significant amount of cells in a variety of tissues during embryogenesis, postnatal development and adulthood. This unintended labelling of cells imposes a serious problem for lineage tracing and mosaic analysis experiments. Importantly, however, we find that reporter constructs differ greatly in their susceptibility to basal CreERT2 activity. While Ai14 and Ai3 easily recombine under basal CreERT2 activity levels, mTmG and R26R-EYFP rarely become activated under these conditions and are therefore better suited for cell tracking experiments.

Les cellules souches pluripotentes (PSC) naïves ont la capacité de réintégrer le développement embryonnaire normal et de produire des fœtus chimériques chez les rongeurs. Cependant, les PSC naïves provenant d'espèces autres que les rongeurs présentent une capacité nettement moins efficace à coloniser les embryons. Actuellement, notre compréhension des mécanismes impliqués dans la formation des chimères est limitée. Le projet avait pour objectif de mieux comprendre les mécanismes impliqués dans la capacité des PSC à coloniser l’embryon pré-implantatoire. Dans un premier temps, nous nous sommes intéressés aux caractéristiques spécifiques des PSC capables de coloniser. Au laboratoire nous avons obtenus des lignées de PSC de lapin et une lignée de PSC de chimpanzé capable de coloniser l’embryon pré-implantatoire de lapin. Nous avons alors séquencé et analysé le transcriptome de ces lignées de PSC de lapin et de chimpanzé pour identifier les caractéristiques moléculaires des PSC capables de coloniser. Ainsi, nous avons montré que les PSC capables de coloniser active la signalisation PI3K, répriment la signalisation Hippo et modulent les voies impliquées dans les interactions cellulaires et la régulation du cytosquelette. Dans un second temps, nous nous sommes intéressés aux mécanismes moléculaires intervenant durant la colonisation de l’embryon par les PSC. Pour cela, nous avons séquencé des embryons de lapin chimériques 48h après injection de PSC de chimpanzé ou de souris capables de coloniser. L’analyse du transcriptome des PSC injectées a révélé une augmentation de la signalisation PI3K/AKT ainsi que des voies de signalisations impliquées dans les jonctions cellulaires, les adhésions cellulaires et les régulations du cytosquelette, suggérant des interactions hôte-PSC injectés. De plus, l’analyse a également révélé qu’une partie de l’épiblaste de l’embryon hôte est composé de PSC injectées sans altération de l’identité de l’hôte. En conclusion, lors de la colonisation, les PSC et les cellules de l’embryon hôte interagissent et communiquent pour harmoniser leur développement
Naïve pluripotent stem cells (PSC) possess the ability to re-enter normal development and generate chimeric fetuses in rodents. However, naïve PSCs from non-rodent species exhibit a significantly less efficient capacity to colonize embryos. Currently, our understanding of the mechanisms involved in chimera formation is limited. The project aimed to decipher these mechanisms. Firstly, we focused on hallmarks of chimeric competent PSCs. In the lab, we obtained chimeric competent PSCs in rabbit and chimpanzee that we analyzed by RNA sequencing analysis to identify the molecular signature of chimeric competent PSCs. We showed that rabbit, chimpanzee as well as mouse PSCs enhance PI3K/AKT signaling, downregulate Hippo signaling and modulate cellular interactions and regulation of cytoskeleton. Secondly, we investigated mechanisms taking place during embryo colonization by PSCs. To this aim, we performed a single-cell RNA sequencing analysis of rabbit embryos colonized by chimpanzee and mouse PSCs. The analysis revealed that injected PSCs increased PI3K/AKT signaling and other signaling pathways involved in cell junction, cell adhesion, and cytoskeleton regulations, suggesting interactions between host embryo cells and injected PSCs. This analysis also revealed that part of the host epiblast is replaced by injected PSCs without any changes of the host cells’ identity. To conclude, during colonization, PSC and cells from the host embryos interact and communicate for efficient colonization