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Genetically engineered mouse models (GEMMs) transformed the study of organismal disease phenotypes but are limited by their lengthy generation in embryonic stem cells. Here, we describe methods for rapid and scalable genome engineering in somatic cells of the liver and pancreas through delivery of CRISPR components into living mice. We introduce the spectrum of genetic tools, delineate viral and nonviral CRISPR delivery strategies and describe a series of applications, ranging from gene editing and cancer modeling to chromosome engineering or CRISPR multiplexing and its spatio-temporal control. Beyond experimental design and execution, the protocol describes quantification of genetic and functional editing outcomes, including sequencing approaches, data analysis and interpretation. Compared to traditional knockout mice, somatic GEMMs face an increased risk for mouse-to-mouse variability because of the higher experimental demands of the procedures. The robust protocols described here will help unleash the full potential of somatic genome manipulation. Depending on the delivery method and envisaged application, the protocol takes 3-5 weeks.

Nitric oxide (NO) exerts both antiatherogenic and proatherogenic effects, but the cellular and molecular mechanisms that contribute to modulation of atherosclerosis by NO are not understood completely. The cGMP-dependent protein kinase I (cGKI) is a potential mediator of NO signaling in vascular smooth muscle cells (SMCs). Postnatal ablation of cGKI selectively in the SMCs of mice reduced atherosclerotic lesion area, demonstrating that smooth muscle cGKI promotes atherogenesis. Cell-fate mapping indicated that cGKI is involved in the development of SMC-derived plaque cells. Activation of endogenous cGKI in primary aortic SMCs resulted in cells with increased levels of proliferation; increased levels of vascular cell adhesion molecule-1, peroxisome proliferator-activated receptor γ, and phosphatidylinositol 3-kinase/Akt signaling; and decreased plasminogen activator inhibitor 1 mRNA, which all are potentially proatherogenic properties. Taken together, these results highlight the pathophysiologic significance of vascular SMCs in atherogenesis and identify a key role for cGKI in the development of atherogenic SMCs in vitro and in vivo . We suggest that activation of smooth muscle cGKI contributes to the proatherogenic effect of NO and that inhibition of cGKI might be a therapeutic option for treating atherosclerosis in humans.

The cGMP-dependent protein kinase type I (cGKI) is a major mediator of NO/cGMP-induced vasorelaxation. Smooth muscle expresses two isoforms of cGKI, cGKIα and cGKIβ, but the specific role of each isoform in vascular smooth muscle cells (VSMCs) is poorly understood. We have used a genetic deletion/rescue strategy to analyze the functional significance of cGKI isoforms in the regulation of the cytosolic Ca 2+ concentration by NO/cGMP in VSMCs. Cultured mouse aortic VSMCs endogenously expressed both cGKIα and cGKIβ. The NO donor diethylamine NONOate (DEA-NO) and the membrane-permeable cGMP analogue 8-bromo-cGMP inhibited noradrenaline-induced Ca 2+ transients in wild-type VSMCs but not in VSMCs genetically deficient for both cGKIα and cGKIβ. The defective Ca 2+ regulation in cGKI-knockout cells could be rescued by transfection of a fusion construct consisting of cGKIα and enhanced green fluorescent protein (EGFP) but not by a cGKIβ-EGFP construct. Fluorescence imaging indicated that the cGKIα-EGFP fusion protein was concentrated in the perinuclear/endoplasmic reticulum region of live VSMCs, whereas the cGKIβ-EGFP protein was more homogeneously distributed in the cytoplasm. These results suggest that one component of NO/cGMP-induced smooth muscle relaxation is the activation of the cGKIα isoform, which decreases the noradrenaline-stimulated cytosolic Ca 2+ level.

Ligand-dependent site-specific recombinases are powerful tools to engineer the mouse genome in specific somatic cell types at selected times during pre- and postnatal development. Current efforts are primarily directed towards increasing the efficiency of this recombination system in mice. We have generated transgenic mouse lines expressing a tamoxifen-activated Cre recombinase, CreER(T2), under the control of the smooth muscle-specific SM22 promoter. Both a randomly integrated transgene [SM-CreER(T2)(tg)] and a transgene that has been "knocked in" into the endogenous SM22 locus [SM-CreER(T2)(ki)] were expressed in smooth muscle-containing tissues. The level of CreER(T2) expression and tamoxifen-induced recombination was lower in SM-CreER(T2)(tg) mice compared with SM-CreER(T2)(ki) mice. Whereas no recombinase activity could be detected in vehicle-treated SM-CreER(T2)(ki) mice, administration of tamoxifen induced the excision of a loxP-flanked reporter transgene in up to 100% of smooth muscle cells. The recombined genome persisted for at least four months after tamoxifen treatment. SM-CreER(T2)(ki) transgenic mice should be useful to study the effects of various somatic mutations in smooth muscle.

Increasing evidence suggests that the second messenger cGMP plays a central role in health and disease, but the downstream mechanisms of cGMP-dependent signaling are not well understood. The cGMP-dependent protein kinase type I (cGKI) is an attractive candidate mediator of cGMP signaling, but the pharmacological analysis of cGKI function is limited by the lack of highly specific cGKI activators and inhibitors. To study the (patho)physiological roles of cGKI in vivo, conventional and conditional cGKI knockout mouse models have been established. Whereas cGKI null mutants have a decreased life span (50% of these mice die before 5 to 6 weeks of age), tissue-specific mutants lacking cGKI selectively in the cerebellum, hippocampus, cardiomyocytes, or smooth muscle cells are fully viable allowing one to perform long-term experiments and to study the cell type-specific role of cGKI in adult animals. The analysis of cGKI-deficient mouse models showed that this protein kinase contributes to many (but not all) effects of cGMP in the cardiovascular, gastrointestinal, and nervous system. For instance, cGKI is involved in the acute regulation of smooth muscle tone and platelet aggregation, but is most likely not essential for basal blood pressure control. Of particular interest, cGKI signaling contributes to long-lasting changes in neuronal plasticity, ranging from axonal pathfinding during embryogenesis to the adaptation of synaptic activity during learning and nociception.

Nitric oxide (NO) modulates a variety of processes in the mammalian brain, but the mechanisms of neuronal NO signaling are poorly understood. In the periphery, many effects of NO are mediated via the generation of the second messenger cyclic guanosine-3′,5′-monophosphate (cGMP) and activation of the cGMP-dependent protein kinase type I (cGKI). However, previous studies suggested that the expression of cGKI in the nervous system is rather restricted, thus, questioning the functional significance of the cGMP/cGKI pathway as a mediator of NO signaling in the brain. Here we have performed a detailed immunohistochemical study to elucidate the distribution of cGKI in the CNS and eye of the mouse. Expression of cGKI protein was detected not only in the previously described areas (cerebellum, hippocampus, dorsomedial hypothalamus) but also in a number of additional regions, such as medulla, subcommissural organ, cerebral cortex, amygdala, habenulae, various hypothalamic regions, olfactory bulb, pituitary gland, and retina. Immunoblotting with isoform-specific antibodies indicated that the cGKIα isoform is prominent in the cerebellum and medulla, whereas the cGKIβ isoform is predominant in the cortex, hippocampus, hypothalamus, and olfactory bulb. Similar levels of the isoforms were detected in the pituitary gland and eye. Thus, it appears that distinct brain regions express distinct cGKI isoforms that signal via distinct pathways. Together, these results improve our understanding of the cellular and molecular mechanisms of NO/cGMP/cGKI signaling and indicate that the distribution and functional relevance of this pathway in the mammalian brain is broader than previously thought.

The cGMP-dependent protein kinase type I (cGKI) is involved in the modulation of neuronal plasticity and learning in the hippocampus and cerebellum. The expression and potential function of cGKI and its isoforms, cGKIα and cGKIβ in other brain regions is not completely resolved. We immunohistochemical study to elucidate the distribution of cGKI protein in the CNS and eye of the mouse. The specificity of the cGKI antibody was controlled by using tissue sections of cGKI knockout mice. Expression of cGKI protein was confirmed in cerebellar Purkinje cells, hippocampus, and dorsomedial hypothalamus. However, we also detected a number of additional cGKI-positive regions, e.g. deep cerebellar nuclei, cerebral cortex, subcommissural organ, various hypothalamic regions, olfactory bulb, pituitary gland, and retina. Immunostaining with isoform-specific antibodies indicated that the cGKIα isoform is prominent in the cerebellum and medulla, whereas cGKIβ is predominant in the cortex, hippocampus, hypothalamus, and olfactory bulb. These results suggest that the distribution and potential function of cGKI in the mammalian brain is broader than previously reported. from 2nd International Conference of cGMP Generators, Effectors and Therapeutic Implications Potsdam, Germany, 10–12 June, 2005