CRISPR-based genome editing of a diurnal rodent, Nile grass rat (Arvicanthis niloticus).

BACKGROUND: Diurnal and nocturnal mammals have evolved distinct pathways to optimize survival for their chronotype-specific lifestyles. Conventional rodent models, being nocturnal, may not sufficiently recapitulate the biology of diurnal humans in health and disease. Although diurnal rodents are potentially advantageous for translational research, until recently, they have not been genetically tractable. The present study aims to address this major limitation by developing experimental procedures necessary for genome editing in a well-established diurnal rodent model, the Nile grass rat (Arvicanthis niloticus). RESULTS: A superovulation protocol was established, which yielded nearly 30 eggs per female grass rat. Fertilized eggs were cultured in a modified rat 1-cell embryo culture medium (mR1ECM), in which grass rat embryos developed from the 1-cell stage into blastocysts. A CRISPR-based approach was then used for gene editing in vivo and in vitro, targeting Retinoic acid-induced 1 (Rai1), the causal gene for Smith-Magenis Syndrome, a neurodevelopmental disorder. The CRISPR reagents were delivered in vivo by electroporation using an improved Genome-editing via Oviductal Nucleic Acids Delivery (i-GONAD) method. The in vivo approach produced several edited founder grass rats with Rai1 null mutations, which showed stable transmission of the targeted allele to the next generation. CRISPR reagents were also microinjected into 2-cell embryos in vitro. Large deletion of the Rai1 gene was confirmed in 70% of the embryos injected, demonstrating high-efficiency genome editing in vitro. CONCLUSION: We have established a set of methods that enabled the first successful CRISPR-based genome editing in Nile grass rats. The methods developed will guide future genome editing of this and other diurnal rodent species, which will promote greater utility of these models in basic and translational research.

CRISPR-based Genome Editing of a Diurnal Rodent, Nile Grass Rat ( Arvicanthis niloticus).

Diurnal and nocturnal mammals have evolved distinct pathways to optimize survival for their chronotype-specific lifestyles. Conventional rodent models, being nocturnal, may not sufficiently recapitulate the biology of diurnal humans in health and disease. Although diurnal rodents are potentially advantageous for translational research, until recently, they have not been genetically tractable. Here, we address this major limitation by demonstrating the first successful CRISPR genome editing of the Nile grass rat ( Arvicanthis niloticus ), a valuable diurnal rodent. We establish methods for superovulation; embryo development, manipulation, and culture; and pregnancy maintenance to guide future genome editing of this and other diurnal rodent species.

Chemerin is resident to vascular tunicas and contributes to vascular tone.

The adipokine chemerin may support blood pressure, evidenced by a fall in mean arterial pressure after whole body antisense oligonucleotide (ASO)-mediated knockdown of chemerin protein in rat models of normal and elevated blood pressure. Although the liver is the greatest contributor of circulating chemerin, liver-specific ASOs that abolished hepatic-derived chemerin did not change blood pressure. Thus, other sites must produce the chemerin that supports blood pressure. We hypothesize that the vasculature is a source of chemerin independent of the liver that supports arterial tone. RNAScope, PCR, Western blot analyses, ASOs, isometric contractility, and radiotelemetry were used in the Dahl salt-sensitive (SS) rat (male and female) on a normal diet. Retinoic acid receptor responder 2 (Rarres2) mRNA was detected in the smooth muscle, adventitia, and perivascular adipose tissue of the thoracic aorta. Chemerin protein was detected immunohistochemically in the endothelium, smooth muscle cells, adventitia, and perivascular adipose tissue. Chemerin colocalized with the vascular smooth muscle marker α-actin and the adipocyte marker perilipin. Importantly, chemerin protein in the thoracic aorta was not reduced when liver-derived chemerin was abolished by a liver-specific ASO against chemerin. Chemerin protein was similarly absent in arteries from a newly created global chemerin knockout in Dahl SS rats. Inhibition of the receptor Chemerin1 by the receptor antagonist CCX832 resulted in the loss of vascular tone that supports potential contributions of chemerin by both perivascular adipose tissue and the media. These data suggest that vessel-derived chemerin may support vascular tone locally through constitutive activation of Chemerin1. This posits chemerin as a potential therapeutic target in blood pressure regulation.NEW & NOTEWORTHY Vascular tunicas synthesizing chemerin is a new finding. Vascular chemerin is independent of hepatic-derived chemerin. Vasculature from both males and females have resident chemerin. Chemerin1 receptor activity supports vascular tone.

Transient receptor potential vanilloid 4 channels are important regulators of parenchymal arteriole dilation and cognitive function.

OBJECTIVE: Hypertension-associated PA dysfunction reduces cerebral perfusion and impairs cognition. This is associated with impaired TRPV4-mediated PA dilation; therefore, we tested the hypothesis that TRPV4 channels are important regulators of cerebral perfusion, PA structure and dilation, and cognition. METHODS: Ten- to twelve-month-old male TRPV4 knockout (WKY-Trpv4em4Mcwi ) and age-matched control WKY rats were studied. Cerebral perfusion was measured by MRI with arterial spin labeling. PA structure and function were assessed using pressure myography and cognitive function using the novel object recognition test. RESULTS: Cerebral perfusion was reduced in the WKY-Trpv4em4Mcwi rats. This was not a result of PA remodeling because TRPV4 deletion did not change PA structure. TRPV4 deletion did not change PA myogenic tone development, but PAs from the WKY-Trpv4em4Mcwi rats had severely blunted endothelium-dependent dilation. The WKY-Trpv4em4Mcwi rats had impaired cognitive function and exhibited depressive-like behavior. The WKY-Trpv4em4Mcwi rats also had increased microglia activation, and increased mRNA expression of GFAP and tumor necrosis factor alpha suggesting increased inflammation. CONCLUSION: Our data indicate that TRPV4 channels play a critical role in cerebral perfusion, PA dilation, cognition, and inflammation. Impaired TRPV4 function in diseases such as hypertension may increase the risk of the development of vascular dementia.