Mouse Chimeras Research Articles

Mouse embryos, chimeras, and embryonal carcinoma stem cells-Reflections on the winding road to gene manipulation.

The relationship of embryonal carcinoma (EC) cells, the stem cells of germ cell- or embryo-derived teratocarcinoma tumors, to early embryonic cells came under intense scrutiny in the early 1970s when mouse chimeras were produced between EC cells and embryos. These chimeras raised tantalizing possibilities and high hopes for different areas of research. The normalization of EC cells by the embryo lent validity to their use as in vitro models for embryogenesis and indicated that they might reveal information about the relationship between malignancy and differentiation. Chimeras also showed the way for the potential introduction of genes, selected in EC cells in vitro, into the germ line of mice. Although EC cells provided material for the elucidation of early embryonic events and stimulated many studies of early molecular differentiation, after years of intense scrutiny,they fell short as the means of genetic manipulation of the germ line, although arguably they pointed the way to the development of embryonic stem (ES) cells that eventually fulfilled this goal.

Characterization of the long-term effects of lethal total body irradiation followed by bone marrow transplantation on the brain of C57BL/6 mice

Purpose Total body irradiation (TBI) followed by bone marrow transplantation (BMT) is used in pre-clinical research to generate mouse chimeras that allow to study the function of a protein specifically on immune cells. Adverse consequences of irradiation on the juvenile body and brain are well described and include general fatigue, neuroinflammation, neurodegeneration and cognitive impairment. Yet, the long-term consequences of TBI/BMT performed on healthy adult mice have been poorly investigated. Material and Methods We developed a robust protocol to achieve near complete bone marrow replacement in mice using 2x550cGy TBI and evaluated the impact of the procedure on their general health, mood disturbances, memory, brain atrophy, neurogenesis, neuroinflammation and blood-brain barrier (BBB) permeability 2 and/or 16 months post-BMT. Results We found a persistent decrease in weight along with long-term impact on locomotion after TBI and BMT. Although the TBI/BMT procedure did not lead to anxiety- or depressive-like behavior 2- or 16-months post-BMT, long-term spatial memory of the irradiated mice was impaired. We also observed radiation-induced impaired neurogenesis and cortical microglia activation 2 months post-BMT. Moreover, higher levels of hippocampal IgG in aged BMT mice suggest an enhanced age-related increase in BBB permeability that could potentially contribute to the observed memory deficit. Conclusions Overall health of the mice did not seem to be majorly impacted by TBI followed by BMT during adulthood. Yet, TBI-induced alterations in the brain and behavior could lead to erroneous conclusions on the function of a protein on immune cells when comparing mouse chimeras with different genetic backgrounds that might display altered susceptibility to radiation-induced damage. Ultimately, the BMT model we here present could also be used to study the related long-term consequences of TBI and BMT seen in patients.

Abstract 125: Elevated Bacterial Fatty Acid Metabolism And Fecal Calorific Values Are Associated With Reduced Diet-induced Adiposity And Weight Gain In Mice

Introduction: Activation of the sympathetic nervous system (SNS) leads to exaggerated immune responses in cardiometabolic conditions. We previously showed that ablation of beta-adrenergic receptors (ADRB) on the bone marrow (BM) hematopoietic cells reduces blood pressure, dampens the immune responses, and modifies gut microbiota, suggesting multisystem host-microbiota interactions. To investigate the role of the microbiota in these complex interactions, we tested the effects of high fat diet (HFD) on the gut microbiota and adiposity in BM mouse chimera characterized by reduced SNS-immune signaling. Methods: Male C5BL/6 wild type (WT) mice were irradiated and reconstituted with BM cells from either the C57BL/6 WT controls or the ADRB 1/2 knock-out mice (KD) to generate BM chimera. Following recovery, mice were fed with a control diet (CNT) or HFD for two weeks ad libitum . Food intake and body weights were measured weekly, and body fat (NMR) and visceral fat pad weights were measured at endpoint. At endpoint, 16S sequencing and metagenomic analyses (QIIME2, PiCRUST2) were performed in cecal samples, and residual fecal calorific values were determined using a simultaneous thermogravimetric analyzer differential calorimeter (TA Instruments). Results: No change in food intake was observed in either group. However, significant increases in body weight, total body fat, and visceral fat weight were observed in the WT compared to KD chimera on HFD ( p<0.05) . HFD elevated gut bacterial Shannon α-diversity (p< 0.05) in the WT but not the KD chimera, and we observed a decrease in g_Faecalibacterium and p_Actinobacteria in WT, and an increase in p_Bacteroidetes and o_Bacteroidales in the KD mice on HDF. Enrichment in fatty acid metabolic pathways and increased fecal calorific values were observed in the KD compared to WT chimera on HFD ( p<0.05) . Conclusion: Reduced SNS-immune signaling is protective against diet-induced weight gain and body fat accumulation. This may in part be mediated by the gut microbiota, reflecting in increased bacterial fatty acid metabolism and elevated residual fecal calorific values, suggesting reduced fat absorption. We propose that selective enrichment of gut bacteria is a potential therapeutic target in diet-induces obesity.

Podocalyxin-Like Protein 1 Regulates Pluripotency through the Cholesterol Biosynthesis Pathway.

Deciphering signaling mechanisms critical for the extended pluripotent stem cell (EPSC) state and primed pluripotency is necessary for understanding embryonic development. Here, a membrane protein, podocalyxin-like protein 1 (PODXL) as being essential for extended and primed pluripotency, is identified. Alteration of PODXL expression levels affects self-renewal, protein expression of c-MYC and telomerase, and induced pluripotent stem cell (iPSC) and EPSC colony formation. PODXL is the first membrane protein reported to regulate de novo cholesterol biosynthesis, and human pluripotent stem cells (hPSCs) are more sensitive to cholesterol depletion than fibroblasts. The addition of exogenous cholesterol fully restores PODXL knockdown-mediated loss of pluripotency. PODXL affects lipid raft dynamics via the regulation of cholesterol. PODXL recruits the RAC1/CDC42/actin network to regulate SREBP1 and SREBP2 maturation and lipid raft dynamics. Single-cell RNA sequencing reveals PODXL overexpression enhanced chimerism between human cells in mouse host embryos (hEPSCs 57%). Interestingly, in the human-mouse chimeras, laminin and collagen signaling-related pathways are dominant in PODXL overexpressing cells. It is concluded that cholesterol regulation via PODXL signaling is critical for ESC/EPSC.

Dealing with symptoms in the general population: lessons learned from the Danish Symptom Cohort

<b>Background:</b> Placental mesenchymal dysplasia (PMD) is a distinct syndrome of unknown aetiology that is associated with significant fetal morbidity and mortality. Intrauterine growth restriction is common, yet, paradoxically, many of the associated fetuses/newborns have been diagnosed with Beckwith-Wiedemann syndrome (BWS). <b>Methods:</b> We report two cases of PMD with high levels of androgenetic (complete paternal uniparental isodisomy) cells in the placenta and document, in one case, a likely androgenetic contribution to the fetus as well. <b>Results:</b> The same haploid paternal complement found in the androgenetic cells was present in coexisting biparental cells, suggesting origin from a single fertilisation event. <b>Conclusions:</b> Preferential allocation of the normal cells into the trophoblast explains the absence of trophoblast overgrowth, a key feature of this syndrome. Interestingly, the distribution of androgenetic cells appears to differ from that reported for artificially created androgenetic mouse chimeras. Androgenetic mosaicism for the first time provides an aetiology for PMD, and may be a novel mechanism for BWS and unexplained intrauterine growth restriction.

Critical Role of Notch-1 in Mechanistic Target of Rapamycin Hyperactivity and Vascular Inflammation in Patients With Takayasu Arteritis.

Takayasu arteritis (TA) is a major type of large vessel vasculitis characterized by progressive inflammation in vascular layers. In our recent study we identified a central role of mechanistic target of rapamycin (mTOR) hyperactivity in proinflammatory T cell differentiation in TA. This study was undertaken to explore potential mechanisms underpinning T cell-intrinsic mTOR hyperactivity and vascular inflammation in TA, with a focus on Notch-1. Notch-1 expression and activity was determined according to Notch-1, activated Notch-1, and HES-1 levels. We detected mTOR activity with intracellular expression of phosphorylated ribosomal protein S6. Differentiation of proinflammatory T cells was analyzed by detecting Th1 and Th17 lineage-determining transcription factors. The function of Notch-1 was evaluated using γ-secretase inhibitor DAPT and gene knockdown using a short hairpin RNA (shRNA) strategy. We performed our translational study using humanized NSG mouse chimeras in which human vasculitis was induced using immune cells from TA patients. CD4+ T cells from TA patients exerted Notch-1high , leading to mTOR hyperactivity and spontaneous maldifferentiation of Th1 cells and Th17 cells. Blockade of Notch-1 using DAPT and Notch-1 shRNA efficiently abrogated mTOR complex 1 (mTORC1) activation and proinflammatory T cell differentiation. Mechanistically, Notch-1 promoted mTOR expression, interacted with mTOR, and was associated with lysosomal localization of mTOR. Accordingly, systemic administration of DAPT and CD4+ T cell-specific gene knockdown of Notch-1 could alleviate vascular inflammation in humanized TA chimeras. Expression of Notch-1 is elevated in CD4+ T cells from TA patients, resulting in mTORC1 hyperactivity and proinflammatory T cell differentiation. Targeting Notch-1 is a promising therapeutic strategy for the clinical management of TA.

Loss of Foxd4 Impacts Neurulation and Cranial Neural Crest Specification During Early Head Development.

The specification of anterior head tissue in the late gastrulation mouse embryo relies on signaling cues from the visceral endoderm and anterior mesendoderm (AME). Genetic loss-of-function studies have pinpointed a critical requirement of LIM homeobox 1 (LHX1) transcription factor in these tissues for the formation of the embryonic head. Transcriptome analysis of embryos with gain-of-function LHX1 activity identified the forkhead box gene, Foxd4, as one downstream target of LHX1 in late-gastrulation E7.75 embryos. Our analysis of single-cell RNA-seq data show Foxd4 is co-expressed with Lhx1 and Foxa2 in the anterior midline tissue of E7.75 mouse embryos, and in the anterior neuroectoderm (ANE) at E8.25 alongside head organizer genes Otx2 and Hesx1. To study the role of Foxd4 during early development we used CRISPR-Cas9 gene editing in mouse embryonic stem cells (mESCs) to generate bi-allelic frameshift mutations in the coding sequence of Foxd4. In an in vitro model of the anterior neural tissues derived from Foxd4-loss of function (LOF) mESCs and extraembryonic endoderm cells, expression of head organizer genes as well as Zic1 and Zic2 was reduced, pointing to a need for FOXD4 in regulating early neuroectoderm development. Mid-gestation mouse chimeras harbouring Foxd4-LOF mESCs displayed craniofacial malformations and neural tube closure defects. Furthermore, our in vitro data showed a loss of FOXD4 impacts the expression of cranial neural crest markers Twist1 and Sox9. Our findings have demonstrated that FOXD4 is essential in the AME and later in the ANE for rostral neural tube closure and neural crest specification during head development.

Fc-Modified Kko: A Novel Therapeutic for Heparin-Induced Thrombocytopenia (HIT), Reversing Both the Thrombocytopenia and Thrombosis

Heparin induced thrombocytopenia (HIT) is an immunogenic prothrombotic disorder caused by antibodies that recognize human platelet factor 4 (PF4) complexed to polyanions. We had previously shown using chimeric constructs of hPF4 and mouse (m) PF4 and chimeras with the related chemokine, neutrophil-activating peptide-2 that there is a single antigenic locus on hPF4 in these complexes to which most HIT antibodies bind. KKO is a mouse monoclonal IgG2b k anti-hPF4/polyanion monoclonal antibody that mimics pathogenic antibodies that induce HIT and provokes thrombosis in a murine model of HIT. We previously established that specific hydrolysis of N-linked glycans in the Fc-region of KKO by endoglycosidase from Streptococcus pyogenes EndoS (Genovis) yields >97% deglycosylation on LC-MS/MS generating DGKKO. This modification has no significant effect on binding to PF4-heparin complexes as shown by ELISA and by dynamic light scattering, but abrogates FcgRIIA-mediated binding and platelet activation, and decreases complement activation as evaluated by flow cytometry. To examine if DGKKO reduces prothrombotic effects, we compared DGKKO with KKO in human microfluidic system lined with human umbilical vein cells (HUVECs) that are then photochemically injured and a murine model involving “HIT mice” (mice that express FcgRIIA and human PF4 and lack mouse PF4). Using the microfluidic system described above and infusing blood from healthy donors with added human PF4 (25 µg/ml) and KKO (50 µg/ml) or HIT IgG from three individuals with SRA-positive HIT (1mg/ml) resulted in increased platelet adherence to the injured endothelium (Figure 1). Addition of DGKKO (50 µg/ml) 15 minutes after addition of HIT antibodies eliminated platelet accumulation (Figure 1). In the HIT murine model, we found that intraperitoneal (IP, 200 µg/mice) or intravenous (IV, 20 µg/mice) DGKKO did not induce thrombocytopenia in HIT mice, but reversed the thrombocytopenia induced by either IP KKO (200 µg/mice) or HIT IgG (1 mg/mice) even when the DGKKO is given 6 hrs after HIT induction (Figure 2A). We used an intravital cremaster laser arteriole injury model in HIT mice to study the efficacy of DGKKO as an antithrombotic agent. We found that unlike KKO that enhances growth of established thrombi in these mice, DGKKO significantly reversed the size of the observed thrombi (Figure 2B). These studies suggest that DGKKO obstructs the HIT antigenic site recognized by HIT antibodies and leads to a reversal of thrombocytopenia and thrombus size. Additional studies are underway to examine if DGKKO can be used as a monotherapy or adjunctive therapy in the murine model of HIT thrombosis. [Display omitted] DisclosuresCines: Rigel: Consultancy; Dova: Consultancy; Treeline: Consultancy; Arch Oncol: Consultancy; Jannsen: Consultancy; Taventa: Consultancy; Principia: Other: Data Safety Monitoring Board.

Chronic infection drives Dnmt3a-loss-of-function clonal hematopoiesis via IFNγ signaling.

Age-related clonal hematopoiesis (CH) is a risk factor for malignancy, cardiovascular disease, and all-cause mortality. Somatic mutations in DNMT3A are drivers of CH, but decades may elapse between the acquisition of a mutation and CH, suggesting that environmental factors contribute to clonal expansion. We tested whether infection provides selective pressure favoring the expansion of Dnmt3a mutant hematopoietic stem cells (HSCs) in mouse chimeras. We created Dnmt3a-mosaic mice by transplanting Dnmt3a-/- and WT HSCs into WT mice and observed the substantial expansion of Dnmt3a-/- HSCs during chronic mycobacterial infection. Injection of recombinant IFNγ alone was sufficient to phenocopy CH by Dnmt3a-/- HSCs upon infection. Transcriptional and epigenetic profiling and functional studies indicate reduced differentiation associated with widespread methylation alterations, and reduced secondary stress-induced apoptosis accounts for Dnmt3a-/- clonal expansion during infection. DNMT3A mutant human HSCs similarly exhibit defective IFNγ-induced differentiation. We thus demonstrate that IFNγ signaling induced during chronic infection can drive DNMT3A-loss-of-function CH.

Direct analysis of brain phenotypes via neural blastocyst complementation.

We provide a protocol for generating forebrain structures in vivo from mouse embryonic stem cells (ESCs) via neural blastocyst complementation (NBC). We developed this protocol for studies of development and function of specific forebrain regions, including the cerebral cortex and hippocampus. We describe a complete workflow, from methods for modifying a given genomic locus in ESCs via CRISPR-Cas9-mediated editing to the generation of mouse chimeras with ESC-reconstituted forebrain regions that can be directly analyzed. The procedure begins with genetic editing of mouse ESCs via CRISPR-Cas9, which can be accomplished in ~4-8 weeks. We provide protocols to achieve fluorescent labeling of ESCs in ~2-3 weeks, which allows tracing of the injected, ESC-derived donor cells in chimeras generated via NBC. Once modified ESCs are ready, NBC chimeras are generated in ~3 weeks via injection of ESCs into genetically programmed blastocysts that are subsequently transferred into pseudo-pregnant fosters. Our in vivo brain organogenesis platform is efficient, allowing functional and systematic analysis of genes and other genomic factors in as little as 3 months, in the context of a whole organism.

John D. Gearhart (1943-2020).

Pioneer of human pluripotent stem cell research

Genetic ablation of bone marrow beta‐adrenergic receptors alters miRNA‐transcriptome networks for microglia activation and inflammation in the paraventricular nucleus of the hypothalamus

Hypertension (HTN) is a preventable condition with complex etiology. Neurogenic origins of HTN such as aberrant signaling in the paraventricular nucleus (PVN) of the hypothalamus remain a high priority for therapeutic interventions, as most anti‐HTN treatments remain ineffective in a significant portion of patients. Discerning the molecular mechanisms underlying neurogenic HTN is critical for understanding how an overactive sympathetic drive contributes to high blood pressure. We generated a novel bone marrow‐specific adrenergic beta 1 and beta 2 knockout mouse chimera (AdrB1.B2 KO mouse) to study how sympathetic drive to the bone, or lack thereof, affects the molecular responses in the PVN. These mice have previously been characterized by dampened systemic immune responses and decreased blood pressure during the active period. Here, loss of sympathetic drive in the AdrB1.B2 KO chimera lead to an overwhelming suppression of transcriptional networks in the PVN that included leukocyte cell adhesion and migration as well as lymphocyte activation and adhesion, and T cell‐ activation and recruitment. Furthermore, transcriptome networks associated with astroglia, microglial, and neuronal function were suppressed in the PVN of AdrB1.B2 KO chimera. Transcriptional networks related to IL‐17a signaling and the renin‐angiotensin system related genes were also suppressed in the PVN of the AdrB1.B2 KO chimera. Using computational predictive tools, we identified a number of miRNAs expected to regulate global transcriptome responses in the PVN. These included miR‐27b3p, miR‐150, miR‐223, and miR‐326, some of which have been implicated in HTN. In addition, using qPCR, we observed a downregulation in the relative expression of miR‐150, miR‐205, miR‐223, miR‐375, miR‐499a, and miR‐27b3p in the PVN of AdrB1.B2 KO chimera, thus in part confirming the results of computational predictive tools. This study identifies novel molecular mechanisms involved in neural‐immune interactions that underlie neurogenic HTN and elucidate new pathways for therapeutic intervention.Support or Funding InformationSupported by AHA grant 14SDG18300010 to JZ and NIH R21AT010192 Award AWD05242 P0104932 to CJM and JZ.

Genetic ablation of bone marrow beta-adrenergic receptors in mice modulates miRNA-transcriptome networks of neuroinflammation in the paraventricular nucleus.

Elucidating molecular pathways regulating neuroimmune communication is critical for therapeutic interventions in conditions characterized by overactive immune responses and dysfunctional autonomic nervous system. We generated a bone marrow-specific adrenergic beta 1 and beta 2 knockout mouse chimera (AdrB1.B2 KO) to determine how sympathetic drive to the bone affects transcripts and miRNAs in the hypothalamic paraventricular nucleus (PVN). This model has previously exhibited a dampened systemic immune response and decreased blood pressure compared with control animals. Reduced sympathetic responsiveness of the bone marrow hematopoietic cells of AdrB1.B2 KO chimera led to suppression of transcriptional networks that included leukocyte cell adhesion and migration and T cell-activation and recruitment. Transcriptome responses related to IL-17a signaling and the renin-angiotensin system were also suppressed in the PVN. Based on the transcriptome response, we next computationally predicted miRNAs in the PVN that may underscore the reduced sympathetic responsiveness of the bone marrow cells. These included miR-27b-3p, miR-150, miR-223-3p, and miR-326. Using real-time PCR, we measured a downregulation in the expression of miR-150-5p, miR-205-5p, miR-223-3p, miR-375-5p, miR-499a-5p, miR-27b-3p, let-7a-5p, and miR-21a-5p in the PVN of AdrB1.B2 KO chimera, confirming computational predictions that these miRNAs are associated with reduced neuro-immune responses and the loss of sympathetic responsiveness in the bone marrow. Intriguingly, directional responses of the miRNA corresponded to mRNAs, suggesting complex temporal or circuit-dependent posttranscriptional control of gene expression in the PVN. This study identifies molecular pathways involved in neural-immune interactions that may act as targets of therapeutic intervention for a dysfunctional autonomic nervous system.

Targeting Mechanistic Target of Rapamycin Complex 1 Restricts Proinflammatory T Cell Differentiation and Ameliorates Takayasu Arteritis.

Takayasu arteritis (TAK) is a progressive autoimmune large vessel vasculitis with infiltration of proinflammatory T cells, with a largely unknown etiology. This study was undertaken to explore the involvement of mechanistic target of rapamycin (mTOR) in proinflammatory T cell differentiation and disease progression in TAK. Ninety-five patients with TAK, 26 patients with small vessel vasculitis, and 40 healthy donors were enrolled. Naive and memory CD4+ T cells were activated with anti-CD3/CD28 beads and analyzed for lineage differentiation. The mTORC1 activity was determined by quantifying intracellular phospho-S6 kinase 1 and phospho-S6 ribosomal protein. Rapamycin and lentiviral regulatory-associated protein of mTOR short hairpin RNA were used to block mTORC1 activity. Human artery-NSG mouse chimeras representing human TAK were established for targeting mTORC1 in disease treatment. TAK CD4+ T cells were selectively prepositioned with hyperactivity of mTORC1 (P < 0.001), resulting in spontaneous maldifferentiation of Th1 and Th17 cells (P < 0.001). Activity of mTORC1high in circulating CD4+ T cells predicted elevated frequencies of proinflammatory T cells and active disease in TAK patients (P < 0.001). Blockade of mTORC1 with rapamycin efficiently abrogated the maldifferentiation of Th1 and Th17 cells (P < 0.01) and ameliorated vasculitis in humanized TAK chimeras (P < 0.001). Inhibition of mTORC1 using RNA interference technology is sufficient to reduce proinflammatory T cell frequencies (P < 0.01) and restrict TAK disease progression in vivo (P < 0.01). Our findings indicate that hyperactivity of mTORC1 is a critical cell-intrinsic mechanism underlying spontaneous maldifferentiation of proinflammatory T cells in TAK patients. Targeting mTORC1 is a promising therapeutic strategy against TAK.

Controlled ploidy reduction of pluripotent 4n cells generates 2n cells during mouse embryo development.

Cells with high ploidy content are common in mammalian extraembryonic and adult tissues. Cell-to-cell fusion generates polyploid cells during mammalian development and tissue regeneration. However, whether increased ploidy can be occasionally tolerated in embryonic lineages still remains largely unknown. Here, we show that pluripotent, fusion-derived tetraploid cells, when injected in a recipient mouse blastocyst, can generate diploid cells upon ploidy reduction. The generated diploid cells form part of the adult tissues in mouse chimeras. Parental chromosomes in pluripotent tetraploid cells are segregated through tripolar mitosis both randomly and nonrandomly and without aneuploidy. Tetraploid-derived diploid cells show a differentiated phenotype. Overall, we discovered an unexpected process of controlled genome reduction in pluripotent tetraploid cells. This mechanism can ultimately generate diploid cells during mouse embryo development and should also be considered for cell fusion-mediated tissue regeneration approaches.

A distinct cardiopharyngeal mesoderm genetic hierarchy establishes antero-posterior patterning of esophagus striated muscle.

In most vertebrates, the upper digestive tract is composed of muscularized jaws linked to the esophagus that permits food ingestion and swallowing. Masticatory and esophagus striated muscles (ESM) share a common cardiopharyngeal mesoderm (CPM) origin, however ESM are unusual among striated muscles as they are established in the absence of a primary skeletal muscle scaffold. Using mouse chimeras, we show that the transcription factors Tbx1 and Isl1 are required cell-autonomously for myogenic specification of ESM progenitors. Further, genetic loss-of-function and pharmacological studies point to MET/HGF signaling for antero-posterior migration of esophagus muscle progenitors, where Hgf ligand is expressed in adjacent smooth muscle cells. These observations highlight the functional relevance of a smooth and striated muscle progenitor dialogue for ESM patterning. Our findings establish a Tbx1-Isl1-Met genetic hierarchy that uniquely regulates esophagus myogenesis and identify distinct genetic signatures that can be used as framework to interpret pathologies arising within CPM derivatives.

Integrase-Mediated Targeted Transgenics Through Pronuclear Microinjection.

Transgenic technology allows a gene of interest to be introduced into the genome of a laboratory animal and provides an extremely powerful tool to dissect the molecular mechanisms of disease. Transgenic mouse models made by microinjection of DNA into zygotic pronuclei, in particular, have been widely used by the genetics community for over 35years. However, up till 5years ago, it remained a rather crude approach: injected sequences randomly insert in multiple copies as concatemers, and they can be mutagenic and have variable, ectopic, or silenced expression depending on the site of integration, a phenomenon called position effects. As a result, multiple lines are required in order to confirm appropriate transgene expression. This can be partially overcome by flanking transgenes with insulator sequences to protect the transgene from influence of surrounding regulatory elements. Large (<300kb) BAC-based transgenic vectors have also been shown to be more resistant to position effects. However, animals carrying extra copies of fairly large regions of the genome could have unpredictable phenotypes.These problems can be overcome by targeting the transgene to a specific chromosomal locus via homologous recombination in embryonic stem (ES) cells. However, this method is significantly more laborious and time consuming, as it involves creation of modified ES cells and mouse chimeras, as well as eventual germline transmission of the transgene.Here, I describe an integrase-based approach, trademarked as "TARGATT™" (target attP), to produce site-specific transgenic mice via pronuclear microinjection, whereby an intact single-copy transgene can be inserted into predetermined chromosomal loci with high efficiency (up to 40%), and faithfully transmitted through generations. This system allows high-level global transgene expression or tissue-specific expression depending on the promoter used, or inducible expression such as induced by tetracycline or doxycycline. Using this approach, site-specific transgenic mice can be generated as fast as in 3months. The technique presented here greatly facilitates murine transgenesis and precise structure/function dissection of mammalian gene function and regulation in vivo.

The Fluctuations of Leukocytes and Circulating Cytokines in Septic Humanized Mice Vary With Outcome.

Sepsis remains a major challenge in translational research given its heterogeneous pathophysiology and the lack of specific therapeutics. The use of humanized mouse chimeras with transplanted human hematopoietic cells may improve the clinical relevance of pre-clinical studies. However, knowledge of the human immuno-inflammatory response during sepsis in humanized mice is scarce; it is unclear how similar or divergent mouse and human-origin immuno-inflammatory responses in sepsis are. In this study, we evaluated the early outcome-dependent immuno-inflammatory response in humanized mice generated in the NSG strain after cecal ligation and puncture (CLP) sepsis. Mice were observed for 32 h post-CLP and were assigned to either predicted-to-die (P-DIE) or predicted-to-survive (P-SUR) groups for retrospective comparisons. Blood samples were collected at baseline, 6 and 24 h, whereas the bone marrow and spleen were collected between 24 and 32 h post-CLP. In comparison to P-SUR, P-DIE humanized mice had a 3-fold higher frequency of human splenic monocytes and their CD80 expression was reduced by 1.3-fold; there was no difference in the HLA-DR expression. Similarly, the expression of CD80 on the bone marrow monocytes from P-DIE mice was decreased by 32% (p < 0.05). Sepsis induced a generalized up-regulation of both human and murine plasma cytokines (TNFα, IL-6, IL-10, IL-8/KC, MCP-1); it was additionally aggravated in P-DIE vs. P-SUR. Human cytokines were strongly overridden by the murine ones (approx. ratio 1:9) but human TNFα was 7-fold higher than mouse TNFα. Interestingly, transplantation of human cells did not influence murine cytokine response in NSG mice, but humanized NSG mice were more susceptible to sepsis in comparison with NSG mice (79 vs. 33% mortality; p < 0.05). In conclusion, our results show that humanized mice reflect selected aspects of human immune responses in sepsis and therefore may be a feasible alternative in preclinical immunotherapy modeling.

CTLA4-Ig Directly Inhibits Osteoclastogenesis by Interfering With Intracellular Calcium Oscillations in Bone Marrow Macrophages.

CTLA4-Ig (cytotoxic T-lymphocyte antigen 4-immunoglobulin; Abatacept) is a biologic drug for rheumatoid arthritis. CTLA4 binds to the CD80/86 complex of antigen-presenting cells and blocks the activation of T cells. Although previous reports showed that CTLA4-Ig directly inhibited osteoclast differentiation, the whole inhibitory mechanism of CTLA4-Ig for osteoclast differentiation is unclear. Bone marrow macrophages (BMMs) from WT mice were cultured with M-CSF and RANKL with or without the recombinant mouse chimera CTLA4-Ig. Intracellular calcium oscillations of BMMs with RANKL were detected by staining with calcium indicator fura-2 immediately after administration of CTLA4-Ig or after one day of treatment. Calcium oscillations were analyzed using Fc receptor gamma- (FcRγ-) deficient BMMs. CTLA4-Ig inhibited osteoclast differentiation and reduced the expression of the nuclear factor of activated T cells NFATc1 in BMMs in vitro. Calcium oscillations in BMMs were suppressed by CTLA4-Ig both immediately after administration and after one day of treatment. CTLA4-Ig did not affect osteoclastogenesis and did not cause remarkable changes in calcium oscillations in FcRγ-deficient BMMs. Finally, to analyze the effect of CTLA4-Ig in vivo, we used an LPS-induced osteolysis model. CTLA4-Ig suppressed LPS-induced bone resorption in WT mice, not in FcRγ-deficient mice. In conclusion, CTLA4-Ig inhibits intracellular calcium oscillations depending on FcRγ and downregulates NFATc1 expression in BMMs. © 2019 American Society for Bone and Mineral Research.

Cell-intrinsic regulation of peripheral memory-phenotype T cell frequencies.

Memory T and B lymphocyte numbers are thought to be regulated by recent and cumulative microbial exposures. We report here that memory-phenotype lymphocyte frequencies in B, CD4 and CD8 T-cells in 3-monthly serial bleeds from healthy young adult humans were relatively stable over a 1-year period, while Plasmablast frequencies were not, suggesting that recent environmental exposures affected steady state levels of recently activated but not of memory lymphocyte subsets. Frequencies of memory B and CD4 T cells were not correlated, suggesting that variation in them was unlikely to be determined by cumulative antigenic exposures. Immunophenotyping of adult siblings showed high concordance in memory, but not of recently activated lymphocyte subsets. To explore the possibility of cell-intrinsic regulation of T cell memory, we screened effector memory-phenotype T cell (TEM) frequencies in common independent inbred mice strains. Using two pairs from these strains that differed predominantly in either CD4 TEM and/or CD8 TEM frequencies, we constructed bi-parental bone marrow chimeras in F1 recipient mice, and found that memory T cell frequencies in recipient mice were determined by donor genotypes. Together, these data suggest cell-autonomous determination of memory T niche size, and suggest mechanisms maintaining immune variability.