Biology subjects

Cowley, D. O.

Publications and source records attributed to Cowley, D. O..

6 recordsLinked to original sources

bioRxiv2026

Lack of canonical PAR4 activation is associated with reduced arterial and venous thrombosis in mice.

BackgroundProtease-activated receptor 4 (PAR4) is the only functional thrombin receptor on mouse platelets. Expression and activation of platelet PAR4 was shown to influence hemostatic plug stability and contributes to thrombosis in different murine arterial and venous thrombosis models. PAR4 activation by thrombin and other serine proteases occurs at the canonical activation site at Arginine (Arg) 59 in mice and Arg47 in humans. If murine PAR4 has functional non-canonical activation sites as shown for other PARs is unknown. ObjectiveTo investigate canonical and potentially non-canonical PAR4 signaling in mice, we generated a mouse model expressing a functional, thrombin-cleavage resistant PAR4 by changing Arg59 to Alanine (Ala) 59 in murine PAR4 (PAR4R59A). PAR4R59A mice were used to assess the impact of impaired canonical (thrombin)-dependent PAR4 signaling on hemostasis and thrombosis in mice. MethodsWe analyzed platelet aggregation, platelet integrin activation and -granule release ex vivo. Hemostasis and thrombosis in PAR4R59A and their control mice was compared using the jugular vein needle puncture injury-induced hemostasis model, and the ferric chloride-induced carotid artery and electrolytic injury-induced femoral vein thrombosis models. ResultsPlatelets of PAR4R59A mice did not response to thrombin but responded normally to PAR4 agonist peptide (PAR4AP) stimulation in aggregation assays. Platelets of PAR4R59A and their control mice exhibited comparable responses to ADP, convulxin or PAR4AP regarding integrin activation and -granular release. PAR4R59A mice exhibited impaired hemostasis in the jugular vein needle puncture model, and were protected from ferric chloride-induced arterial thrombosis and from electrolytic injury induced thrombosis in the femoral vein. ConclusionThe novel PAR4R59A mouse expresses a thrombin-insensitive but still functional PAR4. We propose that the new mouse line will increase the in vivo investigation of canonical PAR4 signaling pathways and may reveal unknown non-canonical PAR4 signaling in different pathologies.

pathology↗

bioRxiv2022

A HUMANIZED FCRN TRANSGENIC MOUSE FOR PRECLINICAL PHARMACOKINETICS STUDIES

Monoclonal antibodies (mAbs) are one of the fastest-growing classes of drugs and have been approved to treat several diseases, including cancers and autoimmune disorders. Preclinical pharmacokinetics studies are performed to determine the therapeutically meaningful dosages and efficacy of candidate drugs. These studies are typically performed in non-human primates; however, using primates is costly and raises ethical considerations. As a result, rodent models that better mimic human-like pharmacokinetics have been generated and remain an area of active investigation. Pharmacokinetic characteristics of a candidate drug, such as half-life, are partly controlled by antibody binding to the human neonatal receptor hFCRN. Due to the abnormally high binding of human antibodies to mouse FCRN, traditional laboratory rodents do not accurately model the pharmacokinetics of human mAbs. In response, humanized rodents expressing hFCRN have been generated. However, these models generally use large inserts randomly integrated into the mouse genome. Here, we report the production and characterization of a CRISPR/Cas9-mediated hFCRN transgenic mouse termed SYNB-hFCRN. Using CRISPR/Cas9-assisted gene targeting, we prepared a strain with a simultaneous knockout of mFcrn and insertion of a hFCRN mini-gene under the control of the endogenous mouse promoter. These mice are healthy and express hFCRN in the appropriate tissues and immune cell subtypes. Pharmacokinetic evaluation of human IgG and adalimumab (Humira(R)) demonstrate hFCRN- mediated protection. These newly generated SYNB-hFCRN mice provide another valuable animal model for use in preclinical pharmacokinetics studies during early drug development. Materials availabilityCryopreserved germline cells from the strains are available from the MMRRC. Dr. Jarnagin will provide sequencing and other evidence supporting strain identity and the plasmids used for constructions.

immunology↗

bioRxiv2022

ROBUST AND EFFICIENT ACTIVE GENETICS GENE CONVERSION IN THE RAT AND MOUSE

The utility of Active Genetic (AG) gene conversion systems in rats and mice holds great promise for facilitating the production of complex strains harboring multiple humanizing genes. The practical application of such systems requires the identification of a robust, reusable, and highly efficient system. By characterizing twenty-eight different promoter and target site pairs we aimed to define the parameters needed to establish an efficient conversion system in male and female rats and mice. Using three specific meiosis prophase I active promoters to drive Cas9 expression. We studied several variables, including the number of Cas9 target sites, the distance between target sites, the cis versus trans configuration in linked pairs, and the effect of Cas9 copy number. In the rat, three of twelve tested configurations provided efficient AG gene conversion in the 22% - 67% range, and four others catalyzed AG in the 0.7-1% range. The rat Ddx4 (Vasa) promoter provides higher AG efficiency than the Sycp1 promoter. In mice, ten of sixteen tested configurations, using the Sycp1 and pSycp1 promoters, provided efficiency in the 0.3% - 3.2% range. In rats, Cas9 expression levels are remarkably well correlated with AG gene conversion efficiency. The rat cis rCyp3A1/rCyp3A2 locus was the most successful configuration, with gene conversion efficiencies of 0.7%-67%. This target site has a special property; the two Cas9 target sites are nearly perfectly homologous in the 100 bases around the gRNA target site. Our findings identify key parameters that improve AG efficiency, including the use of two Cas9 target sites, and efficient promoters that drive high levels of Cas9 expression that are correctly timed during gamete development. These findings also uncover the unexpected benefit of high homology at paired gRNA target sites to promote efficiency. We provide new data to guide future efforts to develop yet further improved AG systems.

genetics↗

bioRxiv2021

Endocardium-to-coronary artery differentiation during heart development and regeneration involves sequential roles of Bmp2 and Cxcl12/Cxcr4.

Regenerating coronary blood vessels has the potential to ameliorate ischemic heart disease, yet there is currently no method of stimulating clinically effective cardiac angiogenesisis. Endocardial cells-- a particularly plastic cell type during development--line the heart lumen and are natural coronary vessel progenitors. Their intrinsic angiogenic potential is lost in adults, but studying the endocardial- to-coronary developmental pathway could identify methods of re-instating this process in diseased hearts. Here, we use a combination of mouse genetics and scRNAseq of lineage-traced endothelial cells to identify novel regulators of endocardial angiogenesis and precisely assess the role of Cxcl12/Cxcr4 signaling. Time-specific lineage tracing demonstrated that endocardial cells differentiated earlier than previously thought, largely at mid-gestation. A new mouse line reporting the activity of Cxcr4 revealed that, despite widespread Cxcl12 and Cxcr4 expression, only a small subset of these coronary endothelial cells activated the receptor, which were mostly in arteries. In accordance with these two findings, Cxcr4 deletion in the endocardial lineage only affected artery formation and only when deleted before mid-gestation. Integrating scRNAseq data of coronary endothelial cells from the endocardial lineage at both mid- and late-gestation identified a transitioning population that was specific to the earlier timepoint that specifically expressed Bmp2. Recombinant Bmp2 stimulated endocardial angiogenesis in an in vitro explant assay and in neonatal mouse hearts upon myocardial infarction. Our data shed light on how understanding the molecular mechanisms underlying endocardial-to-coronary transitions can identify new potential therapeutic targets that could promote revascularization of the injured heart.

developmental biology↗

bioRxiv2020

Baseline and Innate Immune Response Characterization of a Zfp30 Knockout Mouse Strain

Airway neutrophilia is correlated with disease severity in a number of chronic and acute pulmonary diseases, and dysregulation of neutrophil chemotaxis can lead to host tissue damage. The gene Zfp30 was previously identified as a candidate regulator of neutrophil recruitment to the lungs and secretion of CXCL1, a potent neutrophil chemokine, in a genome-wide mapping study using the Collaborative Cross. ZFP30 is a putative transcriptional repressor with a KRAB domain capable of inducing heterochromatin formation. Using a CRISPR-mediated knockout mouse model, we investigated the role that Zfp30 plays in recruitment of neutrophils to the lung using models of allergic airway disease and acute lung injury. We found that the Zfp30 null allele did not affect CXCL1 secretion or neutrophil recruitment to the lungs in response to various innate immune stimuli. Intriguingly, despite the lack of neutrophil phenotype, we found there was a significant reduction in the proportion of live Zfp30 homozygous mutant mice produced from heterozygous matings. This deviation from the expected mendelian inheritance (i.e. transmission ratio distortion) implicates Zfp30 in fertility or embryonic development. Overall, our results indicate that Zfp30 is an essential gene but does not influence neutrophilic inflammation in this particular knockout model.

genetics↗

bioRxiv2020

Elements at the 5' end of Xist harbor SPEN-independent transcriptional antiterminator activity

The Xist lncRNA requires Repeat A, a conserved RNA element located in its 5' end, to induce gene silencing during X-chromosome inactivation. Intriguingly, Repeat A is also required for the production of Xist. While silencing by Repeat A requires the protein SPEN, how Repeat A promotes Xist production remains unclear. We report that in mouse embryonic stem cells, expression of a transgene comprising the first two kilobases of Xist (Xist-2kb) causes transcriptional readthrough of multiple downstream polyadenylation sequences. Readthrough required Repeat A and the ~750 nucleotides downstream but did not require SPEN. Despite associating with SPEN and chromatin, Xist-2kb did not robustly silence transcription, whereas a transgene comprising Xists first 5.5 kilobases robustly silenced transcription and read through its polyadenylation sequence. Longer, spliced Xist transgenes also induced robust silencing yet terminated efficiently. Thus, in contexts examined here, Xist requires sequence elements beyond its first two kilobases to robustly silence transcription, and the 5' end of Xist harbors SPEN-independent transcriptional antiterminator activity that can repress proximal cleavage and polyadenylation. In endogenous contexts, this antiterminator activity may help produce full-length Xist RNA while rendering the Xist locus resistant to silencing by the same repressive complexes that the lncRNA recruits to other genes.

molecular biology↗