Neuromuscular and cardiovascular diseases are active areas for application of CRISPR-Cas9 targeting to query the role of targeted mutations in recapitulating or correcting features of the analogous human genetic defects in vivo in live animals. Recently, porcine zygote injection with Cas9/sgRNA resulted in the generation of a piglet with muscular dystrophy (1). The piglet exhibited degenerative skeletal and cardiac muscle, potentially modeling symptoms of human muscular dystrophy (1). The CRISPR-Cas9 system has also been used to generate a rat model of Duchenne muscular dystrophy (2). Mutated rats exhibited a decline in muscle strength, and the emergence of degenerative phenotypes in skeletal muscle and heart. The mutations were heritable by the next generation, and F1 male rats exhibited similar phenotypes in their skeletal muscles (2). In contrast, Exon 23 deletion by CRISPR-Cas9 in the dystrophin-deficient mdx mouse resulted in expression of the dystrophin gene, partial recovery of functional dystrophin protein in skeletal myofibers and cardiac muscle, and significant enhancement of muscle force. (3) Others too have demonstrated that genome editing partially restores dystrophin expression in mice with muscular dystrophy (4, 5). Since many neuromuscular disorders results in gait and heart abnormalities, study of the heart and motor function in conditional mutant animals remains paramount to their validation as disease or therapeutic models. DigiGait and ECGenie , therefore, are efficient methods to validate the causal links between genotype and phenotype needed in pre-clinical models, and to identify potential off-target activity. The ECGenie non-invasively monitors the heart in awake mice, from day 1 of life through death. It easily informs regarding arrhythmia, conduction defects, and heart failure. DigiGait provides metrics of strength, mobility, balance, and coordination in animal models of disorders that affect the ability of subjects to walk and run normally. DigiGait, for example, reports metrics of strength and force generation in animals with muscular-skeletal problems, including models of limb girdle muscular dystrophy, Duchenne muscular dystrophy, ossification of Achilles tendon (6), and Friedreich's Ataxia (FA). DigiGait technology recently identified significant gait disturbances in the KIKO mouse model of FA providing phenotypic measures related to Friedreich's ataxia clinical endpoints that could be used to test effectiveness of potential drug therapy (7). DigiGait , moreover, would quantify dystonia in mice with homozygous mutation of the VPS16 gene accomplished through CRISPR-Cas9 genome-editing (8). The ECGenie is now widely used to non-invasively record the ECG in awake mice. It has demonstrated, for example, autonomic nervous system abnormalities in modulation of the heart in mdx mice (9), as well the d-sarcoglycan deficient hamster model of limb-girdle muscular dystrophy (10). It would most certainly report the electrocardiographic disturbances in mice with PRKAG2 cardiac syndrome (11), and it would most certainly demonstrate the effectiveness of CRISPR/Cas9 genome editing to treat the PRKAG2 cardiac syndrome in mice, to show the power of the technology to characterize dominant inherited cardiac diseases resulting by disrupting disease-causing mutations. Advances in genome-editing techniques such as CRISPR/Cas9 will bolster the generation of genetically modified live animals that will more closely resemble the disease-causing mutations and recapitulate pathological features of human conditions. Mouse Specifics, Inc. non-invasive gait and heart monitoring technologies will rapidly characterize, early in life , the many models of neurodegenerative, neuromuscular, and cardiovascular diseases resulting from targeted gene editing methods and technologies. References: Yu HH et al. Porcine Zygote Injection with Cas9/sgRNA Results in DMD-Modified Pig with Muscle Dystrophy. Int J Mol Sci. 2016;17(10). Nakamura K et al. Generation of muscular dystrophy model rats with a CRISPR/Cas system. Sci Rep. 2014;4:5635. Nelson CE et al. In vivo genome editing improves muscle function in a mouse model of Duchenne muscular dystrophy. Science. 2016;351(6271):403-7. Long C, et al. Postnatal genome editing partially restores dystrophin expression in a mouse model of muscular dystrophy. Science. 2016;351(6271):400-3. Xu L et al. CRISPR-mediated Genome Editing Restores Dystrophin Expression and Function in mdx Mice. Mol Ther. 2016;24(3):564-9. Suzuki H et al. Gene targeting of the transcription factor Mohawk in rats causes heterotopic ossification of Achilles tendon via failed tenogenesis. Proc Natl Acad Sci U S A. 2016 ;113(28):7840-5 McMackin MZ et al. Neurobehavioral deficits in the KIKO mouse model of Friedreich's ataxia. Behav Brain Res. 2017;316:183-188. Cai X et al. Homozygous mutation of VPS16 gene is responsible for an autosomal recessive adolescent-onset primary dystonia. Sci Rep. 2016;6:25834. Chu V, et al. Electrocardiographic findings in mdx mice: a cardiac phenotype of Duchenne muscular dystrophy. Muscle Nerve. 2002; 26(4):513-9. Hampton TG, et al. Developmental Changes in the ECG of a Hamster Model of Muscular Dystrophy and Heart Failure. Front Pharmacol. 2012;3:80. Xie C et al. Genome editing with CRISPR/Cas9 in postnatal mice corrects PRKAG2 cardiac syndrome. Cell Res. 2016; 26(10):1099-1111.

Either you are intrigued with the hysteria regarding clown sightings, or eager to learn the latest about the presidential election. In either case, please read on. Though clowns hope to make us laugh, they sometimes instill fear; fear and laughter, stress and joy, greatly affect our cardiovascular health. Many adults, moreover, are finding the presidential election in the United States entertaining, yet stressful. Benoit Poisson from the University of Montreal provides a beautiful treatise on how the development of neurohormonal circuits underlies our basic emotions – stress, anger, sorrow and happiness (1). Emotions enable an individual to constantly modulate the cardiovascular system to the physical and social environment. Mouse Specifics, Inc. developed the ECGenie to study how neurohormonal circuits affect the modulation of the heart of laboratory animals, providing a window to study their emotions in models of human disorders. Whether lab mice experience emotions in the same manner of humans is debatable, but of course we know that mice in pain have elevated heart rate and reduced heart rate variability (HRV), reflecting the same response that humans have to unalleviated pain. Fearful mice also have elevated heart rate and lower HRV, just like people who find themselves on a turbulent airplane. Mice provided enrichment in their environments reduced heart rate and higher HRV, akin to humans enriched by a viewing a comical movie or listening to Debussy. While the grimace scale for lab animals has been validated for pain assessment (2), a metric for “smiling” in “happy” lab animals has not yet been put forth. Laughing and weeping have profound affects on HRV (3). Heart rate variability increases, therefore, could serve as a surrogate marker for happiness in mice, as it is well established that interventions and agents that reduce pain and bring joy to humans increase HRV in animals. Angelman syndrome (AS), a neurodevelopmental disorder, is characterized by easily provoked smiling and laughter. Though pathological laughing or crying reflect complex aberrant neurobiological phenomena, it is interesting that Angelman patients have dysfunction in the autonomic nervous system (ANS). It might be interesting to monitor the heart of mice genetically modified to model AS. The ECGenie , for example, and companion EzCG analyses software, would report HRV in newborn UBE3A mutant mice, to see whether they too have aberrant ANS dysfunction. Please visit our exhibit at SfN (booth 237) to learn how the ECGenie can provide new information about the cardiac and nervous systems in your animal models. Learn how the DigiGait Imaging System can describe motor function in your Angelman syndrome models or any other genetic disorders that affect the heart and nervous system of infants and children. Share with us some serious discussion about your research goals. And we promise to bring out a smile! References: 1. Poisson, P; Systemic biopsychological perspective of basic emotions. Sante Ment Que. 2015 Fall;40(3):223-44. 2. Leung V et al. Real-time application of the Rat Grimace Scale as a welfare refinement in laboratory rats. Sci Rep. 2016 ;6:31667. 3. Sakuragi S et al. . Effects of laughing and weeping on mood and heart rate variability. J Physiol Anthropol Appl Human Sci. 2002; 21:159-65.

Canadian Association for Laboratory Animal Science Annual Meeting Much like the Toronto Blue Jay’s performances, CALAS 2016 was a grand slam. A great venue, great colleagues, and cutting edge research all contributed to the big win for the 55 th Annual Symposium held at the Fairmont Hotel in Toronto, earlier this baseball season. Now half way through the season, the Blue Jays are tied with our own Boston Red Sox and only a few games back from 1 st place, and plans for the next CALAS symposium in Calgary for June 2017 are already in progress. We are all winners with such dedication to doing our best. The enthusiastic responses from our presentation about the heart rate in mice, throughout life and death, were greatly appreciated. Thank you one and all for attending the presentation, and for the many great questions and discussion stemming from the topic. The ECGenie not only reports the ECG and all of the ECG interval durations in interesting mouse models of cardiovascular diseases, but also describes heart rate changes occurring in a wide range of disorders, including pain, Zika virus, and Rett syndrome. We have extended our patented technology to now describe how anesthetics affect the heart rate of awake animals as they succumb to anesthesia, and how pain may be reflected in changes in heart rate variability. The excellent presentations and posters at CALAS 2016 are too many to comment on all. Near and dear to our own research and instrumentation, I was impressed with several presentations. Ovidiu Jumanca from the Institut de Recherches Cliniques de Montréal presented data to show that the sulfamethoxazole /trimethoprim antibiotic combination was more efficacious when administered in a gel rather than via a water bottle. Dr. Jumanca and his colleagues hypothesized that the antibiotic diffused in MediGel [ ClearH2O ] would not precipitate as it does in water, thereby providing more consistent delivery. They found that after 15 days of treatment plasma levels of the drugs reached superior and protective levels in the MediGel treated animals compared to those that received the drugs via their drinking water. Chelsea Schuster from the laboratory of Dr. Daniel Pang presented beautiful data about the benefits of external warming of laboratory rats during isoflurane anesthesia. She reported that recovery to sternal recumbency was fastest in animals that were pre-warmed prior to anesthesia. Furthermore, even limited warming of the animal after anesthesia helped maintain body temperature and accelerated recovery. These observations are of interest to our instrumentation enhancements, as our ECGenie is now able to monitor laboratory animals not only at baseline, but also during anesthesia and during recovery. We have similarly noted heart rate changes during anesthesia that are dependent on whether or not the animal is warmed during anesthesia. Moreover, the recovery of the heart rate to baseline is dependent on the presence of warmth during anesthesia. We are further inspired by another study presented by Dr. Pang’s group, presented by Jessie Chisholm. There is a lot of discussion about euthanasia and the best methods for sacrificing laboratory animals. Jessie presented data to show the relationship between loss of consciousness and heart rate during CO 2 euthanasia in rats, concluding that bradycardia occurs prior to the loss of consciousness in rats euthanized with CO 2 or CO 2 + O 2 . The ECGenie coupled with EzCG analysis software are able to discriminate ECG changes specific to the vitality of a sedated rodent maintained on anesthesia from those specific to the expiring rodent whose cardiovascular stability is capitulating to the lethality of CO 2 inhalation. Mouse Specifics, Inc. was a proud sponsor of the annual CALAS symposium, given that we share the CALAS dedication to providing high quality training and educational resources to animal care professionals across Canada, and throughout the world.

Mouse Specifics, Inc. is a proud sponsor of the FSH Society Boston Basketball Tournament March 21, 2016, at the TD Garden. The FSH Society is a nonprofit, patient-driven organization supporting research and education for facioscapulohumeral muscular dystrophy (FSHD), one of the most prevalent forms of muscular dystrophy. Founded in 1991 by two individuals with FSHD, Steve Jacobsen and Daniel Perez, the FSH Society is the world’s largest and most progressive grassroots network of patients, families, clinicians and research activists. Among the most prevalent of the muscular dystrophies, FSHD affects an estimated 870,000 people worldwide. Basic research in animal models is crucial to understanding and treating FSHD. Mouse Specifics, Inc. technology has been used in discoveries in numerous preclinical models of neuromuscular disorders, including Duchenne muscular dystrophy, Limb Girdle Muscular Dystrophy, and Spinal Muscular Atrophy. Please consider donating to the FSH to fund important research! Come to TD Garden to watch the Mouse Specifics team dazzle with our basketball skills on March 21! Any FSH-funded researchers with a mouse model relevant to FSHD are welcome to send us animals for DigiGait Imaging or ECGenie monitoring , as a courtesy to help advance your research.

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