The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) system is an adaptive immune system in bacteria and archaea that has recently been exploited for genome engineering. Mutant mice can be generated in one step through direct delivery of the CRISPR/Cas9 components into a mouse zygote. Although the technology is robust, delivery remains a bottleneck, as it involves manual injection of the components into the pronuclei or the cytoplasm of mouse zygotes, which is technically demanding and inherently low throughput. To overcome this limitation, we employed electroporation as a means to deliver the CRISPR/Cas9 components, including Cas9 messenger RNA, single-guide RNA, and donor oligonucleotide, into mouse zygotes and recovered live mice with targeted nonhomologous end joining and homology-directed repair mutations with high efficiency. Our results demonstrate that mice carrying CRISPR/Cas9-mediated targeted mutations can be obtained with high efficiency by zygote electroporation.

Abstract-Aldosterone classically promotes unidirectional transepithelial sodium transport, thereby regulating blood volume and blood pressure. Recently, both clinical and experimental studies have suggested additional, direct roles for aldosterone in the cardiovascular system. To evaluate aldosterone activation of cardiomyocyte mineralocorticoid receptors, transgenic mice overexpressing 11␤-hydroxysteroid dehydrogenase type 2 in cardiomyocytes were generated using the mouse ␣-myosin heavy chain promoter. This enzyme converts glucocorticoids to receptor-inactive metabolites, allowing aldosterone occupancy of cardiomyocyte mineralocorticoid receptors. Transgenic mice were normotensive but spontaneously developed cardiac hypertrophy, fibrosis, and heart failure and died prematurely on a normal salt diet. Eplerenone, a selective aldosterone blocker, ameliorated this phenotype. These studies confirm the deleterious consequences of inappropriate activation of cardiomyocyte mineralocorticoid receptors by aldosterone and reveal a tonic inhibitory role of glucocorticoids in preventing such outcomes under physiological conditions. In addition, these data support the hypothesis that aldosterone blockade may provide additional therapeutic benefit in the treatment of heart failure.

Joint pathology following DMM surgery in mice is associated with progressive mechanical allodynia. ADAMTS-5 null mice are resistant to DMM-induced OA-like lesions and to the associated mechanical allodynia.

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Mouse embryonic stem cells (mESCs) cultured in the presence of LIF occupy a ground state with highly active pluripotency-associated transcriptional and epigenetic circuitry. However, ground state pluripotency in some inbred strain backgrounds is unstable in the absence of ERK1/2 and GSK3 inhibition. Using an unbiased genetic approach, we dissect the basis of this divergent response to extracellular cues by profiling gene expression and chromatin accessibility in 170 genetically heterogeneous mESCs. We map thousands of loci affecting chromatin accessibility and/or transcript abundance, including 10 QTL hotspots where genetic variation at a single locus coordinates the regulation of genes throughout the genome. For one hotspot, we identify a single enhancer variant $10 kb upstream of Lifr associated with chromatin accessibility and mediating a cascade of molecular events affecting pluripotency. We validate causation through reciprocal allele swaps, demonstrating the functional consequences of noncoding variation in gene regulatory networks that stabilize pluripotent states in vitro. ll

Mitochondrial creatine kinases form octameric structures composed of four active and stable dimers. Octamer formation has been postulated to occur via interaction of the charged amino acids in the N-terminal peptide of the mature enzyme. We altered codons for charged amino acids in the N-terminal region of mature sarcomeric mitochondrial creatine kinase (sMtCK) to those encoding neutral amino acids. Transfection of normal sMtCK cDNA or those with the mutations R42G, E43G/H45G, and K46G into rat neonatal cardiomyocytes resulted in enzymatically active sMtCK expression in all. After hypoosmotic treatment of isolated mitochondria, mitochondrial inner membrane-associated and soluble sMtCK from the intermembranous space were measured. The R42G and E43G/H45G double mutation caused destabilization of the octameric structure of sMtCK and a profound reduction in binding of sMtCK to the inner mitochondrial membrane. The other mutant sMtCK proteins had modest reductions in binding. Creatine-stimulated respiration was markedly reduced in mitochondria isolated from cells transfected with the R42G mutant cDNA as compared with those transfected with normal sMtCK cDNA. We conclude that neutralization of charges in N-terminal peptide resulted in destabilization of octamer structure of sMtCK. Thus, charged amino acids at the N-terminal moiety of mature sMtCK are essential for octamer formation, binding of sMtCK with inner mitochondrial membrane, and coupling of sMtCK to oxidative phosphorylation. Sarcomeric mitochondrial creatine kinase (sMtCK)1 is a member of a gene family of four homologous CK genes (1-4). The M-CK and B-CK genes encode soluble, cytoplasmic homodimeric enzymes necessary for ATP production from creatine phosphate at sites of high energy utilization, such as the contractile apparatus. These cytosolic CKs share about 85% similarity to each other in a particular species, and each has 77-91% homology among vertebrate species. Two additional genes encode different mitochondrial CKs, which are localized on the outer surface of the inner mitochondrial membrane (IMM) (2, 3). sMtCK expression is restricted to heart and skeletal muscle, but ubiquitous MtCK is present in many tissues, especially brain and smooth muscle. Mammalian MtCKs are only 60 -65% similar to cytosolic CKs and about 80% homologous to each other, but sMtCKs share 95% identity across species. This suggests that, in addition to enzymatic functions, ubiquitous MtCK and sMtCK may have slightly different structural roles requiring sequence conservation across species.We previously isolated and characterized cDNA clones encoding human (2, 3) and rat (7) MtCKs. The rat sMtCK cDNA coding region consists of 1260 nucleotides, and the first 117 bp encode the transit peptide, responsible for transport of de novo synthesized precursor sMtCK from cytosol into mitochondria. During this translocation of pre-sMtCK to the intermembranous space, the transit peptide is proteolytically removed, allowing formation of the active, mature subunit (2, 4).Both MtCK proteins can f...

Porcine cells devoid of three major carbohydrate xenoantigens, αGal, Neu5GC, and SDa (TKO) exhibit markedly reduced binding of human natural antibodies. Therefore, it is anticipated that TKO pigs will be better donors for human xenotransplantation. However, previous studies on TKO pigs using old world monkeys (OWMs) have been disappointing because of higher anti‐TKO pig antibodies in OWMs than humans. Here, we show that long‐term survival of renal xenografts from TKO pigs that express additional human transgenes (hTGs) can be achieved in cynomolgus monkeys. Kidney xenografts from TKO‐hTG pigs were transplanted into eight cynomolgus recipients without pre‐screening for low anti‐pig antibody titers. Two recipients of TKO‐hTG xenografts with low expression of human complement regulatory proteins (CRPs) (TKO‐A) survived for 2 and 61 days, whereas six recipients of TKO‐hTG xenografts with high CRP expression (TKO‐B) survived for 15, 20, 71, 135, 265, and 316 days. Prolonged CD4+T cell depletion and low anti‐pig antibody titers, which were previously reported important for long‐term survival of αGal knock‐out (GTKO) xenografts, were not always required for long‐term survival of TKO‐hTG renal xenografts. This study indicates that OWMs such as cynomolgus monkeys can be used as a relevant model for clinical application of xenotransplantation using TKO pigs.

tions in PCSK9 cause hypercholesterolemia in humans ( 1-4 ), whereas loss-of-function mutations in humans lead to a reduction in LDL cholesterol and a marked decrease in the risk of coronary heart disease ( 5-7 ). PCSK9 is a member of the subtilisin serine protease family and the proteinase K subfamily. The protein is synthesized as a proprotein that autocatalytically cleaves to generate mature protein ( 8 ). PCSK9 protein is secreted as a complex containing cleaved N-terminal prodomain, which remains associated with the catalytic domain ( 8 ). The catalytic activity is required for PCSK9 maturation and secretion ( 9 ) but appears not to be essential for the reduction of LDLR (LDL receptor) by secreted PCSK9 ( 10 ).PCSK9 is predominantly expressed in liver, intestine, and to a lesser extent, in kidney ( 8 ). PCSK9 gene expression is regulated by cholesterol via sterol regulatory element-binding protein (SREBP) pathways ( 11,12 ). In mice, PCSK9 gene expression is downregulated by dietary cholesterol and dramatically upregulated by hepatic overexpression of the transactivation domain of SREBP-1a and SREBP-2 ( 12 ). Deletion of PCSK9 in mice leads to elevated liver LDLR protein (but not mRNA) and to reduced plasma total cholesterol, mainly HDL cholesterol ( 13 ). Furthermore, PCSK9 defi ciency increases LDL clearance and enhances the response to statin ( 13 ). Knockdown of PCSK9 by antisense approach also decreases total cholesterol in high-fat-fed mice in an LDLR dependent manner ( 14 ). Hepatic overexpression of murine or human PCSK9 by adenovirus ( 9, 15, 16 ) or transgene ( 17 ) results in the reduction of liver LDLR protein and elevation of LDL cholesterol. Transgenic mice overexpressing PCSK9 in liver accumulated PCSK9 to a level of 100-400 g/ml in plasma and reduced liver LDLR protein in wild-type mice that are parabiotically Abstract Proprotein convertase subtilisin/kexin type 9 (PCSK9) is predominantly expressed in liver and regulates cholesterol metabolism by down regulating liver LDL receptor (LDLR) proteins. Here we report transgenic overexpression of human PCSK9 in kidney increased plasma levels of PCSK9 and subsequently led to a dramatic reduction in liver LDLR proteins. The regulation of LDLR by PCSK9 displayed tissue specifi city, with liver being the most responsive tissue. Even though the PCSK9 transgene was highly expressed in kidney, LDLR proteins were suppressed to a lower extent in this tissue than in liver. Adrenal LDLR proteins were not regulated by elevated plasma PCSK9. hPCSK9 transgene expression and subsequent reduction of liver LDLR led to increases in plasma total cholesterol, LDL cholesterol, and ApoB, which were further increased by a highfat, high-cholesterol diet. We also observed that the size distribution of hPCSK9 in transgenic mouse plasma was heterogeneous. In chow-fed mice, the majority of PCSK9 proteins were in free forms; however, feeding a high-fat, high-cholesterol diet resulted in a shift of hPCSK9 distribution toward larger complexes. PCSK9 distribution in human plasma...