Abstract-Familial hypertrophic cardiomyopathy (FHC) is an inherited autosomal dominant disease caused by mutations in sarcomeric proteins. Among these, mutations that affect myosin binding protein-C (MyBP-C), an abundant component of the thick filaments, account for 20% to 30% of all mutations linked to FHC. However, the mechanisms by which MyBP-C mutations cause disease and the function of MyBP-C are not well understood. Therefore, to assess deficits due to elimination of MyBP-C, we used gene targeting to produce a knockout mouse that lacks MyBP-C in the heart. Knockout mice were produced by deletion of exons 3 to 10 from the endogenous cardiac (c) MyBP-C gene in murine embryonic stem (ES) cells and subsequent breeding of chimeric founder mice to obtain mice heterozygous (ϩ/Ϫ) and homozygous (Ϫ/Ϫ) for the knockout allele. Wild-type (ϩ/ϩ), cMyBP-C ϩ/Ϫ , and cMyBP-C Ϫ/Ϫ mice were born in accordance with Mendelian inheritance ratios, survived into adulthood, and were fertile. Western blot analyses confirmed that cMyBP-C was absent in hearts of homozygous knockout mice. Whereas cMyBP-C ϩ/Ϫ mice were indistinguishable from wild-type littermates, cMyBP-C Ϫ/Ϫ mice exhibited significant cardiac hypertrophy. Cardiac function, assessed using 2-dimensionally guided M-mode echocardiography, showed significantly depressed indices of diastolic and systolic function only in cMyBP-C Ϫ/Ϫ mice. Ca 2ϩ sensitivity of tension, measured in single skinned myocytes, was reduced in cMyBP-C Ϫ/Ϫ but not cMyBP-C ϩ/Ϫ mice. These results establish that cMyBP-C is not essential for cardiac development but that the absence of cMyBP-C results in profound cardiac hypertrophy and impaired contractile function. Key Words: myosin binding protein-C Ⅲ heart Ⅲ myocardium Ⅲ gene knockout Ⅲ sarcomeric proteins M yosin binding protein-C (MyBP-C), also known as C-protein, 1 is a thick filament accessory protein that is present in nearly all vertebrate striated muscles but whose function is unknown. Nonetheless, there is compelling evidence to suggest that MyBP-C is a significant determinant of muscle contractile properties. In particular, cardiac MyBP-C (cMyBP-C) is a target for phosphorylation in response to various inotropic stimuli, including sympathetic stimuli that effect trisphosphorylation of cMyBP-C via cAMP-dependent protein kinase (PKA). 2 In addition, mutations of the cMyBP-C gene are a leading cause of familial hypertrophic cardiomyopathy (FHC), 3 an inherited disorder linked to mutations in cardiac contractile proteins (for review, see Bonne et al 4 and Seidman and Seidman 5 ).However, despite clues suggesting the importance of cMyBP-C to cardiac health, the function of cMyBP-C has remained enigmatic. For instance, although numerous studies have investigated effects of PKA on cardiac contractility (eg, Strang et al 6 and Patel et al 7 ), the role, if any, of cMyBP-C in mediating contractile responses to PKA has been difficult to discern. 8 -10 Similarly, the mechanisms by which cMyBP-C mutations affect cardiac function are not well understo...

Endothelin receptor B (Ednrb) plays a critical role in the development of melanocytes and neurons and glia of the enteric nervous system. These distinct neural crest-derived cell types express Ednrb and share the property of intercalating into tissues, such as the intestine whose muscle precursor cells also express Ednrb. Such widespread Ednrb expression has been a significant obstacle in establishing precise roles for Ednrb in development. We describe here the production of an Ednrb allele floxed at exon 3 and its use in excising the receptor from mouse neural crest cells by use of Cre-recombinase driven by the Wnt1 promoter. Mice born with neural crest-specific excision of Ednrb possess aganglionic colon, lack trunk pigmentation, and die within five weeks due to megacolon. Ednrb receptor expression in these animals is absent only in the neural crest but present in surrounding smooth muscle cells. The absence of Ednrb from crest cells also results in a compensatory upregulation of Ednrb expression in other cells within the gut. We conclude that Ednrb loss only in neural crest cells is sufficient to produce the Hirschsprungs disease phenotype observed with genomic Ednrb mutations.Endothelins make up a family of 21 amino acid peptides, ET-1, ET-2 and ET-3. . These peptides bind to two distinct G-protein-coupled receptors, ET-A and ET-B. The affinity of ET-A is greater for ET-1 and ET-2 than for ET-3 whereas the affinity of ET-B is the same for the three peptides. ET-1, the most ubiquitous, is produced by endothelial cells in the vasculature, epithelial cells in the kidney, heart cells, and some neurons, while ET-2 and ET-3 are both found in the gastrointestinal tract. Both ET-A and ET-B receptors are found in a variety of tissues and activate a number of different effectors. Both receptors are prominent in the vasculature where ET-A mediates vasoconstriction by activating smooth muscle contraction and ET-B receptors produce vasodilation by release of nitric oxide from the endothelial cells under certain conditions (Schneider et al., 2007). Both receptors can also act in mitogen signaling and are known to play a role in cancer (Grant et al., 2006;Lahav et al., 1996).The endothelin receptors have discrete roles during embryonic development by influencing the fate of different neural crest cell populations. The cephalic neural crest invades the pharyngeal arches and gives rise to the bones and cartilage of the facial skeleton, dermis, and smooth muscle of the great arteries. The ET-A receptor appears in the cephalic neural crest cells as they emigrate from the neural tube while the ligand ET-1 is produced by the non-crest The ET-B receptor is required for normal development of the vagal and truncal neural crest (Heanue and Pachnis, 2007). The vagal neural crest cells give rise to enteric neurons and truncal neural crest are precursors for sympathetic and sensory neurons, and melanocytes. Global knockouts or naturally-occurring mutations of the ET-B receptor result in the absence of neural crest-derived neuro...

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Plasma-derived polyclonal antibody therapeutics, such as intravenous immunoglobulin, have multiple drawbacks, including low potency, impurities, insufficient supply, and batch-to-batch variation. Here we describe a microfluidics and molecular genomics strategy for capturing diverse mammalian antibody repertoires to create recombinant multivalent hyperimmune globulins. Our method generates thousands-diverse mixtures of recombinant antibodies, enriched for specificity and activity against therapeutic targets. Each hyperimmune globulin product comprised thousands to tens of thousands of antibodies derived from convalescent or vaccinated human donors, or immunized mice. Using this approach, we generated hyperimmune globulins with potent neutralizing activity against Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) in under three months, Fc-engineered hyperimmune globulins specific for Zika virus that lacked antibody-dependent enhancement of disease, and hyperimmune globulins specific for lung pathogens present in patients with primary immune deficiency. To address the limitations of rabbit-derived anti-thymocyte globulin (ATG), we generated a recombinant human version and demonstrated its efficacy in mice against graft-versus-host disease.

The jumonji (jmj) gene plays important roles in multiple organ development in mouse, including cardiovascular development. Since JMJ is expressed widely during mouse development, it is essential that conditional knockout approaches be employed to ablate JMJ in a tissue-specific manner to identify the cell lineage specific roles of JMJ. In this report, we describe the establishment of a jmj conditional null allele in mice by generating a loxP-flanked (floxed) jmj allele, which allows the in vivo ablation of jmj via Cre recombinase-mediated deletion. Gene targeting was used to introduce loxP sites flanking exon 3 of the jmj allele to mouse embryonic stem cells. Our results indicate that the jmj floxed allele converts to a null allele in a heart-specific manner when embryos homozygous for the floxed jmj allele and carrying the alpha-myosin heavy chain promoter-Cre transgene were analyzed by Southern and Northern blot analyses. Therefore, this mouse line harboring the conditional jmj null allele will provide a valuable tool for deciphering the tissue and cell lineage specific roles of JMJ.