Microinjection of CRISPR/Cas9 requires the availability of zygotes that implies animal breeding, superovulation schemes, and embryo collection. Vitrification of zygotes may allow having ready-to-use ...embryos and to temporally dissociate the workload of embryo production from microinjection. In this study, fresh (F group) or vitrified (V group) zygotes were microinjected with CRISPR/Cas9 system to test the hypothesis that vitrified zygotes could be a suitable source of embryos for microinjection. In Experiment 1 (in vitro evaluation), B6D2F1/J zygotes were microinjected and cultured until blastocyst stage. Embryo survival and cleavage rates after microinjection were similar between groups (~50% and ~80% respectively; P = NS). Development rate was significantly higher for F than V group (55.0% vs. 32.6%, respectively; P<0.05). Mutation rate did not show statistical differences among groups (P = NS). In Experiment 2 (in vivo evaluation), C57BL/6J zygotes were microinjected and transferred to recipient females. Embryo survival was significantly lower in fresh than in vitrified zygotes (49.2% vs. 62.7%, respectively; P<0.05). Cleavage rate did not show statistical differences (~70%; P = NS). Pregnancy rate (70.0% vs. 58.3%) and birth rate (11.9% vs. 11.2%) were not different between groups (F vs. V group; P = NS). Offspring mutation rate was higher for F than V group, in both heterodimer analysis (73.7% vs. 33.3%, respectively; P = 0.015) and Sanger sequencing (89.5% vs. 41.7%, respectively; P = 0.006). In conclusion, vitrified-warmed zygotes present a viable alternative source for CRISPR/Cas9 microinjection when the production of fresh embryos is impeded by limited technical support. The possibility of zygote cryobanking to perform microinjection sessions on demand seems to be a suitable alternative to avoid the breeding and maintenance of animals all over the year, enhancing the implementation of CRISPR technology.
Recent biotechnological applications in the field of clinical oncology led to the identification of new biomarkers as molecular targets of cancer, and to broad developments in the field of ...personalized medicine. Aptamers are oligonucleotides (ssDNA or RNA) that are selected to specifically recognize a molecular target with high affinity and specificity. Based on this, new horizons for their use as molecular imaging probes are being explored. The objective of this work was to evaluate the Sgc8-c aptamer conjugated with Alexa Fluor 647 fluorophore as an imaging probe in a colon tumor xenograft mouse model, with potential application in molecular imaging. In this study, the LS174T cell line was used to induce colorectal adenocarcinoma in nude mice. After confirmation of PTK7 overexpression by immunohistochemistry, in vivo studies were performed. Pharmacokinetic, in vivo and ex vivo biodistribution imaging, and a competition assay were evaluated by fluorescence imaging. In vivo visualization of the probe in the tumors was assessed two hours after aptamer probe administration, exhibiting excellent tumor-to-background ratios in biodistribution studies and high specificity in the competition test. Our results demonstrated the functionality of Scg8-c as an imaging probe for colon cancer, with potential clinical applications.
CCDC28B (coiled-coil domain-containing protein 28B) was identified as a modifier in the ciliopathy Bardet-Biedl syndrome (BBS). Our previous work in cells and zebrafish showed that CCDC28B plays a ...role regulating cilia length in a mechanism that is not completely understood. Here we report the generation of a Ccdc28b mutant mouse using CRISPR/Cas9 (Ccdc28b mut). Depletion of CCDC28B resulted in a mild phenotype. Ccdc28b mut animals i) do not present clear structural cilia affectation, although we did observe mild defects in cilia density and cilia length in some tissues, ii) reproduce normally, and iii) do not develop retinal degeneration or obesity, two hallmark features of reported BBS murine models. In contrast, Ccdc28b mut mice did show clear social interaction defects as well as stereotypical behaviors. This finding is indeed relevant regarding CCDC28B as a modifier of BBS since behavioral phenotypes have been documented in BBS. Overall, this work reports a novel mouse model that will be key to continue evaluating genetic interactions in BBS, deciphering the contribution of CCDC28B to modulate the presentation of BBS phenotypes. In addition, our data underscores a novel link between CCDC28B and behavioral defects, providing a novel opportunity to further our understanding of the genetic, cellular, and molecular basis of these complex phenotypes.
High lipid content of oocytes and embryos in domestic animals is one of the well-known factors associated with poor cryosurvival. Herein, we wanted to determine whether the use of delipidated estrous ...sheep serum during in vitro maturation (IVM) of ovine oocytes reduces the cytoplasmic lipid droplets content and improves embryo development and cryotolerance after vitrification. Cumulus oocytes complexes (COCs) were matured in vitro for 24 h in medium supplemented with whole or delipidated estrous sheep serum prior to vitrification. Neutral lipid present in lipid droplets of COCs, cleavage rate, embryo development rate on Day 6 and Day 8, and hatching rate on Day 8, were compared among experimental groups. Endoplasmic reticulum stress genes were evaluated in in vitro matured COCs under different lipid conditions prior to vitrification. The lipid droplets' content (mean fluorescence intensity) of oocytes cultured with IVM media supplemented with delipidated serum was lower than COCs matured with whole serum (7.6 ± 1.7 vs. 22.8 ± 5.0 arbitrary units, respectively; P< 0.05). Despite IVM treatment, oocytes subjected to vitrification showed impaired competence compared with the non-vitrified groups (P<0.05). No significant differences in embryo production were observed in non-vitrified COCs after maturation in delipidated or whole serum (33.4±4.9 vs 31.9 ±4.2). COCs matured in delipidated serum and subjected to vitrification showed increased expression of ATF4, ATF6, GRP78, and CHOP10 genes (ER stress markers). Collectively, our results demonstrate that although supplementation of IVM medium with delipidated estrous sheep serum reduces the presence of cytoplasmic lipid droplets in oocytes after maturation, oocyte cryotolerance is not improved. Notably, the expression of genes associated with the unfolded protein response (UPR) was increased in COCs, with fewer lipid droplets subjected to vitrification, suggesting that oocyte cryopreservation is associated with ER stress and activation of adaptive responses.
SPATS1 (spermatogenesis-associated, serine-rich 1) is an evolutionarily conserved, testis-specific protein that is differentially expressed during rat male meiotic prophase. Some reports have ...suggested a link between SPATS1 underexpression/mutation and human pathologies such as male infertility and testicular cancer. Given the absence of functional studies, we generated a Spats1 loss-of-function mouse model using CRISPR/Cas9 technology. The phenotypic analysis showed no overt phenotype in Spats1-/- mice, with both males and females being fertile. Flow cytometry and histological analyses did not show differences in the testicular content and histology between WT and knockout mice. Moreover, no significant differences in sperm concentration, motility, and morphology, were observed between WT and KO mice. These results were obtained both for young adults and for aged animals. Besides, although an involvement of SPATS1 in the Wnt signaling pathway has been suggested, we did not detect changes in the expression levels of typical Wnt pathway-target genes in mutant individuals. Thus, albeit Spats1 alteration might be a risk factor for male testicular health, we hereby show that this gene is not individually essential for male fertility and spermatogenesis in mouse.
Echinococcus granulosus
is a cestode parasite which causes cystic echinococcosis disease. Previously we observed that vaccination with
E. granulosus
antigens from human hydatid cyst fluid (HCF) ...significantly inhibits colon cancer growth. In the present work, we evaluate the anti-tumor immune response induced by human HCF against LL/2 lung cancer in mice. HCF vaccination protected from tumor growth, both in prophylactic and therapeutic settings, and significantly increased mouse survival compared to control mice. Considering that tumor-associated carbohydrate antigens are expressed in
E. granulosus,
we oxidized terminal carbohydrates in HCF with sodium periodate. This treatment abrogates the anti-tumor activity induced by HCF vaccination. We found that HCF vaccination-induced IgG antibodies that recognize LL/2 tumor cells by flow cytometry. An antigen-specific immune response is induced with HCF vaccination in the tumor-draining lymph nodes and spleen characterized by the production of IL-5 and, in less extent, IFNɣ. In the tumor microenvironment, we found that NK1.1 positive cells from HCF-treated mice showed higher expression of CD69 than control mice ones, indicating a higher level of activation. When we depleted these cells by administrating the NK-specific antibody NK1.1, a significantly decreased survival was observed in HCF-induced mice, suggesting that NK1.1
+
cells mediate the anti-tumor protection induced by HCF. These results suggest that HCF can evoke an integrated anti-tumor immune response involving both, the innate and adaptive components, and provide novel insights into the understanding of the intricate relationship between HCF vaccination and tumor growth
.
Inflammation is an important process for epithelial barrier protection but when uncontrolled, it can also lead to tissue damage. The nuclear factor-kappa light chain enhancer of activated B cells ...(NF-κB) signaling pathway is particularly relevant in the intestine, as it seems to play a dual role. Whereas NF-κB protects intestinal epithelium against various noxious stimuli, the same pathway mediates intestinal inflammatory diseases by inducing pro-inflammatory gene expression. The availability of appropriate in vitro models of the intestinal epithelium is crucial for further understanding the contribution of NF-κB in physiological and pathological processes and advancing in the development of drugs and therapies against gut diseases. Here we established, characterized, and validated three-dimensional cultures of intestinal organoids obtained from biopsies of NF-κB-RE-Luc mice. The NF-κB-RE-Luc intestinal organoids derived from different intestine regions recreated the cellular composition of the tissue and showed a reporter responsiveness similar to the in vivo murine model. When stimulated with TNF-α, jejunum-derived NF-κB-RE-Luc-reporter organoids, provided a useful model to evaluate the anti-inflammatory effects of natural and synthetic compounds. These reporter organoids are valuable tools to explore the epithelial TNF-α-induced NF-κB contribution in the small intestine, being a reliable alternative method while helping to reduce the use of laboratory animals for experimentation.
Emerging evidence suggests that immune receptors may participate in many aging-related processes such as energy metabolism, inflammation, and cognitive decline. CD300f, a TREM2-like lipid-sensing ...immune receptor, is an exceptional receptor as it integrates activating and inhibitory cell-signaling pathways that modulate inflammation, efferocytosis, and microglial metabolic fitness. We hypothesize that CD300f can regulate systemic aging-related processes and ultimately healthy lifespan. We closely followed several cohorts of two strains of CD300f−/− and WT mice of both sexes for 30 months and observed an important reduction in lifespan and healthspan in knockout mice. This was associated with systemic inflammaging, increased cognitive decline, reduced brain glucose uptake observed by 18FDG PET scans, enrichment in microglial aging/neurodegeneration phenotypes, proteostasis alterations, senescence, increased frailty, and sex-dependent systemic metabolic changes. Moreover, the absence of CD300f altered macrophage immunometabolic phenotype. Taken together, we provide strong evidence suggesting that myeloid cell CD300f immune receptor contributes to healthy aging.
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•CD300f−/− mice show reduced lifespan, but no specific cause of death is identified•This is associated with inflammaging, senescence, frailty, and cognitive decline•CD300f−/− mice exhibit microglial aging states and sex-dependent metabolic changes•Absence of CD300f reduces macrophage immunometabolic fitness in a sex-dependent way
Evans et al. report that the absence of CD300f myeloid cell immune receptor in male and female mice induces a reduced lifespan. Animals display diverse aged-related conditions such as inflammaging, frailty, cognitive decline, brain senescence markers, microglial aging phenotypes, and reduced macrophage metabolic fitness, as well as sex-dependent metabolic alterations.
Interferon gamma (IFN-γ) may be potential adjuvant immunotherapy for COVID-19 patients. In this work, we assessed gene expression profiles associated with the IFN-γ pathway in response to SARS-CoV-2 ...infection. Employing a case-control study from SARS-CoV-2-positive and -negative patients, we identified IFN-γ-associated pathways to be enriched in positive patients. Bioinformatics analyses showed upregulation of MAP2K6, CBL, RUNX3, STAT1, and JAK2 in COVID-19-positive vs. -negative patients. A positive correlation was observed between STAT1/JAK2, which varied alongside the patient’s viral load. Expression of MX1, MX2, ISG15, and OAS1 (four well-known IFN-stimulated genes (ISGs)) displayed upregulation in COVID-19-positive vs. -negative patients. Integrative analyses showcased higher levels of ISGs, which were associated with increased viral load and STAT1/JAK2 expression. Confirmation of ISGs up-regulation was performed in vitro using the A549 lung cell line treated with Poly (I:C), a synthetic analog of viral double-stranded RNA; and in different pulmonary human cell lines and ferret tracheal biopsies infected with SARS-CoV-2. A pre-clinical murine model of Coronavirus infection confirmed findings displaying increased ISGs in the liver and lungs from infected mice. Altogether, these results demonstrate the role of IFN-γ and ISGs in response to SARS-CoV-2 infection, highlighting alternative druggable targets that can boost the host response.
Rodents used in biomedical research are maintained behind barriers to exclude microbial contaminants. Several check points have to be monitored to eliminate the potential of introducing adventitious ...agents into the facility. Microbiological monitoring of a mouse facility environment enables to evaluate the efficiency of sanitization and cleaning procedures, air quality, and technician good practices. At our SPF mouse facility, we implemented an environmental microbiological monitoring program based in sedimentation and swabbing, inexpensive and easy to use methods. The aim of this work was to evaluate the results and the efficiency of the monitoring program after seven years. The median for bacteria and fungi counts in the SPF sampled areas was ≤2 CFU/2 h for settle plates and <1 CFU per swabbing plate, satisfying the requirements for grade C of the EU-GMP, with some modifications. The environmental monitoring program was useful to detect early warning of problems and enabled us to define a safe range of microbiological counts. In addition, SPF status defined for our mice was maintained throughout this study, confirmed by our HM program. This work could encourage directors and technicians of other mouse facilities in Latin America and rest of the world to implement this kind of program.
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