A random mutation capture assay to detect genomic point mutations in mouse tissue

Herein, a detailed protocol for a random mutation capture (RMC) assay to measure nuclear point mutation frequency in mouse tissue is described. This protocol is a simplified version of the original method developed for human tissue that is easier to perform, yet retains a high sensitivity of detection. In contrast to assays relying on phenotypic selection of reporter genes in transgenic mice, the RMC assay allows direct detection of mutations in endogenous genes in any mouse strain. Measuring mutation frequency within an intron of a transcribed gene, we show this assay to be highly reproducible. We analyzed mutation frequencies from the liver tissue of animals with a mutation within the intrinsic exonuclease domains of the two major DNA polymerases, δ and ε. These mice exhibited significantly higher mutation frequencies than did wild-type animals. A comparison with a previous analysis of these genotypes in Big Blue mice revealed the RMC assay to be more sensitive than the Big Blue assay for this application. As RMC does not require analysis of a particular gene, simultaneous analysis of mutation frequency at multiple genetic loci is feasible. This assay provides a versatile alternative to transgenic mouse models for the study of mutagenesis in vivo

Glutamate Receptor Interacting Protein 1 Mediates Platelet Adhesion and Thrombus Formation.

Thrombosis-associated pathologies, such as myocardial infarction and stroke, are major causes of morbidity and mortality worldwide. Because platelets are necessary for hemostasis and thrombosis, platelet directed therapies must balance inhibiting platelet function with bleeding risk. Glutamate receptor interacting protein 1 (GRIP1) is a large scaffolding protein that localizes and organizes interacting proteins in other cells, such as neurons. We have investigated the role of GRIP1 in platelet function to determine its role as a molecular scaffold in thrombus formation. Platelet-specific GRIP1-/- mice were used to determine the role of GRIP1 in platelets. GRIP1-/- mice had normal platelet counts, but a prolonged bleeding time and delayed thrombus formation in a FeCl3-induced vessel injury model. In vitro stimulation of WT and GRIP1-/- platelets with multiple agonists showed no difference in platelet activation. However, in vivo platelet rolling velocity after endothelial stimulation was significantly greater in GRIP1-/- platelets compared to WT platelets, indicating a potential platelet adhesion defect. Mass spectrometry analysis of GRIP1 platelet immunoprecipitation revealed enrichment of GRIP1 binding to GPIb-IX complex proteins. Western blots confirmed the mass spectrometry findings that GRIP1 interacts with GPIbα, GPIbβ, and 14-3-3. Additionally, in resting GRIP1-/- platelets, GPIbα and 14-3-3 have increased interaction compared to WT platelets. GRIP1 interactions with the GPIb-IX binding complex are necessary for normal platelet adhesion to a stimulated endothelium

<i>In vitro</i> platelet activation is normal in GRIP1^-/- platelets.

<p>A) WT and GRIP1^-/- platelets have similar agonist-induced activation. Washed platelets were incubated with control buffer, 50 ng/mL convulxin, or 0.5 U/mL thrombin and P-selectin expression was determined by flow cytometry (N = 4; ± S.E.M., NS by students T-test between WT and GRIP1^-/-). B) PF4 release from stimulated WT and GRIP1^-/- platelets. Washed platelets were stimulated with thrombin or 2-meADP for 10 min. and PF4 release measured by ELISA (N = 8; ± S.E.M, N.S. by students—test). C) Washed WT and GRIP1^-/- platelets were thrombin-stimulated and ATP release was measured (N = 8; ± S.E.M, N.S. by students T-test). D) Washed WT and GRIP1^-/- platelets have similar GPIIb/IIIa activation. Platelets were stimulated with thrombin or 2-meADP for 10 min. Activated GPIIb/IIIa expression was measured by JON/A antibody binding. (N = 4; ± S.E.M, N.S. by students T-test). E) PRP from WT or GRIP1^-/- mice was incubated with either PE or APC labeled anti-CD9 antibody. PE and APC labeled PRP from mice of the same genotype was then mixed. Control buffer, thrombin or 2-meADP were added and incubated with orbital shaking at 37°C for 15 minutes. Double positive (APC and PE) platelet aggregates were quantified by flow cytometry. GRIP1^-/- platelets stimulated with low dose thrombin had a trend to fewer platelet aggregates compared to WT (N = 6–8). WT and GRIP1^-/- platelets stimulated with 2-meADP had similar aggregation.</p

Proteins highly enriched in mass spectrometry analysis with known roles in platelet function (light gray indicates interact with GPIb complex, darker shading indicates other functional platelet protein interactions).

<p>Proteins highly enriched in mass spectrometry analysis with known roles in platelet function (light gray indicates interact with GPIb complex, darker shading indicates other functional platelet protein interactions).</p

GRIP1^-/- mice have hemostasis and thrombosis defects.

<p>A) WT and platelet GRIP1^-/- mouse bleeding time. Percentage of patent vessels after 3 mm tail tip amputation (N = 25–37; P < 0.05 for Kaplan-Meier curve). B) Platelet GRIP1^-/- mice have reduced thrombus size. FeCl₃-induced mesenteric artery thrombosis. Fluorescent thrombus burden expressed as percentage of vessel area (N = 11–17; ± S.E.M., *P < 0.05 by students T-test). C) Representative image of thrombus formation in WT and platelet GRIP1^-/- mice 10 min. after injury (dashed lines represent vessel edges, magnification 20X).</p

GRIP1^-/- platelets do not have decreased velocity in ionophore-stimulated blood vessels.

<p>A) GRIP1^-/- platelets do not have a decreased velocity in an ionophore-stimulated pinna vessel. WT murine pinna vessels were stimulated with A23187 and the change in fluorescent-labeled WT and GRIP1^-/- platelet velocity determined over time. As a control, platelets were treated with GPIbα blocking antibody (N = 4; *P < 0.05 by students T-test GRIP1^-/- vs WT). B) Representative images of WT and GRIP1^-/- platelets before and 6 minutes after vessel ionophore treatment (smaller, rounder platelets are rolling (arrow), magnification 20X). C) GRIP1^-/- platelets do not have decreased velocity in an ionophore-stimulated mesenteric vessel (N = 4 ± S.E.M; *P < 0.05 by students T-test GRIP1^-/- vs WT). D) Representative images of WT and GRIP1^-/- platelets before and after ionophore treatment in mesenteric arterioles (smaller, rounder platelets are rolling (arrow), magnification 20X). E) GRIP1^-/- platelets have a trend toward reduced vWF adhesion in vitro. Platelets in whole blood from WT and GRIP1^-/- mice were labeled with a fluorescent antibody. Blood was then pumped over a mouse vWF-coated MatTek chamber. Platelet adhesion was determined at multiple time points (N = 4–6 per time point, P < 0.1 at 15 sec).</p

GRIP1 interacts with the GPIb-IX complex.

<p>A) GRIP1 protein interactions in platelets. GRIP1 was immunoprecipitated from WT and GRIP1^-/- (negative control) platelets and the precipitate analyzed by mass spectrometry. Proteins were assigned by Panther into GO functional groups. B) GPIbα and GPIbβ surface expression is similar in WT and GRIP1^-/- platelets. GPIbα and GPIbβ surface expression was determined by flow cytometry (N = 8–13, ± S.E.M; NS by students T-test).</p

Platelets express GRIP1.

<p>A) Human platelets express GRIP1 (representative immunoblot with mouse brain as positive control). B) PF4-Cre⁺ GRIP1^fl/fl mice have reduced platelet GRIP1 expression. Immunoprecipitation of WT and GRIP1^-/- mouse platelets using anti-GRIP1 antibody and immunoblot for GRIP1. C) Representative histogram of GRIP1 expression in WT and GRIP1^-/- mouse platelets by intracellular flow cytometry. D) WT and GRIP1^-/- mouse platelets have similar platelet counts. (± S.E.M., NS by students T-test). E) WT and GRIP1^-/- platelets have similar morphology (representative electron microscopy of resting platelets).</p