ACD can configure probes for the various manual and automated assays for INS for RNAscope Assay, or for Basescope Assay compatible for your species of interest.
Cell Death and Disease (2018) 9:1118
2018 Nov 02
Moon S, Im SK, Kim N, Youn H, Park UH, Kim JY, Kim AR, An SJ, Kim JH, Sun W, Hwang JT, Kim EJ, Um SJ.
PMID: - | DOI: 10.1038/s41419-018-1171-z
Nat Commun.
2018 Oct 02
Roccio M, Perny M, Ealy M, Widmer HR, Heller S, Senn P.
PMID: 30279445 | DOI: 10.1038/s41467-018-06334-7
Sensory hair cells located in the organ of Corti are essential for cochlear mechanosensation. Their loss is irreversible in humans resulting in permanent hearing loss. The development of therapeutic interventions for hearing loss requires fundamental knowledge about similarities and potential differences between animal models and human development as well as the establishment of human cell based-assays. Here we analyze gene and protein expression of the developing human inner ear in a temporal window spanning from week 8 to 12 post conception, when cochlear hair cells become specified. Utilizing surface markers for the cochlear prosensory domain, namely EPCAM and CD271, we purify postmitotic hair cell progenitors that, when placed in culture in three-dimensional organoids, regain proliferative potential and eventually differentiate to hair cell-like cells in vitro. These results provide a foundation for comparative studies with otic cells generated from human pluripotent stem cells and for establishing novel platforms for drug validation.
J Control Release.
2019 Feb 13
Stephens CJ, Lauron EJ, Kashentseva E, Lu ZH, Yokoyama WM, Curiel DT.
PMID: 30771412 | DOI: 10.1016/j.jconrel.2019.02.009
Hemophilia B (HB) is a life-threatening inherited disease caused by mutations in the FIX gene, leading to reduced protein function and abnormal blood clotting. Due to its monogenic nature, HB is one of the primary targets for gene therapy. Indeed, successful correction of HB has been shown in clinical trials using gene therapy approaches. However, application of these strategies to non-adult patients is limited due to high cell turnover as young patients develop, resulting in vector dilution and subsequent loss of therapeutic expression. Gene editing can potentially overcome this issue by permanently inserting the corrective gene. Integration allows replication of the therapeutic transgene at every cell division and can avoid issues associated with vector dilution. In this study, we explored adenovirus as a platform for corrective CRISPR/Cas9-mediated gene knock-in. We determined as a proof-of-principle that adenoviral delivery of CRISPR/Cas9 is capable of corrective gene addition, leading to long-term augmentation of FIX activity and phenotypic correction in a murine model of juvenile HB. While we found on-target error-free integration in all examined samples, some mice also contained mutations at the integration target site. Additionally, we detected adaptive immune responses against the vector and Cas9 nuclease. Overall, our findings show that the adenovirus platform is suitable for gene insertion in juveniles with inherited disease, suggesting this approach may be applicable to other diseases.
JCI Insight.
2019 Mar 05
Bailey KE, MacGowan GA, Tual-Chalot S, Phillips L, Mohun TJ, Henderson DJ, Arthur HM, Bamforth SD, Phillips HM.
PMID: 30835717 | DOI: 10.1172/jci.insight.125172
Sarcomeric disarray is a hallmark of gene mutations in patients with Hypertrophic Cardiomyopathy (HCM). However, it is unknown when detrimental sarcomeric changes first occur and whether they originate in the developing embryonic heart. Furthermore, Rho Kinase (ROCK) is a serine threonine protein kinase that is critical for regulating the function of several sarcomeric proteins and therefore, our aim was to determine if disruption of ROCK signalling during the earliest stages of heart development would disrupt the integrity of sarcomeres altering heart development and function. Using a mouse model in which the function of ROCK is specifically disrupted in embryonic cardiomyocytes we demonstrate a progressive cardiomyopathy that first appeared as sarcomeric disarray during cardiogenesis. This led to abnormalities in the structure of embryonic ventricular wall and compensatory cardiomyocyte hypertrophy during foetal development. This sarcomeric disruption and hypertrophy persisted throughout adult life, triggering left ventricular concentric hypertrophy with systolic dysfunction, and re-activation of foetal gene expression and cardiac fibrosis, all typical features of HCM. Taken together, our findings establish a novel mechanism for the developmental origin of the sarcomeric phenotype of HCM and suggest that variants in the ROCK genes or disruption of ROCK signalling could, in part, contribute to its pathogenesis.
Journal of Crohn's and Colitis
2019 Apr 11
Wyss A, Weder B, Mamie C, Rogler G, Hausmann M, Scharl M, Gonzalez Alvarado MN, Spalinger MR, Raselli T, Misselwitz B, Van Haaften WT, Dijkstra G, Sailer AW, Wagner CA, Imenez Silva PH, Tosevski V, Leibl S.
PMID: - | DOI: 10.1093/ecco-jcc/jjz039
Abstract
Intestinal fibrosis and stenosis are common complications of Crohn’s disease [CD], frequently requiring surgery. Anti-inflammatory strategies can only partially prevent fibrosis; hence, anti-fibrotic therapies remain an unmet clinical need. Oxysterols are oxidised cholesterol derivatives with important roles in various biological processes. The enzyme cholesterol 25-hydroxylase [CH25H] converts cholesterol to 25-hydroxycholesterol [25-HC], which modulates immune responses and oxidative stress. In human intestinal samples from CD patients, we found a strong correlation of CH25H mRNA expression with the expression of fibrosis markers. We demonstrate reduced intestinal fibrosis in mice deficient for the CH25H enzyme, using the sodium dextran sulphate [DSS]-induced chronic colitis model. Additionally, using a heterotopic transplantation model of intestinal fibrosis, we demonstrate reduced collagen deposition and lower concentrations of hydroxyproline in CH25H knockouts. In the heterotopic transplant model, CH25H was expressed in fibroblasts. Taken together, our findings indicate an involvement of oxysterol synthesis in the pathogenesis of intestinal fibrosis.
Journal of Investigative Dermatology
2019 May 23
Sola-Carvajal A, Revêchon G, Helgadottir H, Whisenant D, Hagblom R, Döhla J, Katajisto P, Brodin D, Fagerström-Billai F, Viceconte N, Eriksson M.
PMID: 31128203 | DOI: 10.1016/j.jid.2019.05.005
Hutchinson-Gilford progeria syndrome (HGPS) is the result of a defective form of the lamin A protein called progerin. While progerin is known to disrupt the properties of the nuclear lamina, the underlying mechanisms responsible for the pathophysiology of HGPS remain less clear. Previous studies in our laboratory have shown that progerin expression in murine epidermal basal cells results in impaired stratification and halted development of the skin. Stratification and differentiation of the epidermis is regulated by asymmetric stem cell division. Here, we show that expression of progerin impairs the ability of stem cells to maintain tissue homeostasis as a result of altered cell division. Quantification of basal skin cells showed an increase in symmetric cell division that correlated with progerin accumulation in HGPS mice. Investigation of the mechanisms underlying this phenomenon revealed a putative role of Wnt/β-catenin signaling. Further analysis suggested an alteration in the nuclear translocation of β-catenin involving the inner and outer nuclear membrane proteins, emerin and nesprin-2. Taken together, our results suggest a direct involvement of progerin in the transmission of Wnt signaling and normal stem cell division. These insights into the molecular mechanisms of progerin may help develop new treatment strategies for HGPS
J Vis Exp.
2018 Aug 14
Anderson CM, Laeremans A, Wang Xm, Wu X, Zhang B, Doolittle E, Kim J, Li N, Pimentel HXY, Park P, Ma XJ.
PMID: 30176002 | DOI: 10.3791/58097
Because precision medicine is highly dependent on the accurate detection of biomarkers, there is an increasing need for standardized and robust technologies that measure RNA biomarkers in situ in clinical specimens. While grind-and-bind assays like RNAseq and quantitative RT-PCR enable highly sensitive gene expression measurements, they also require RNA extraction and thus prevent valuable expression analysis within the morphological tissue context. The in situ hybridization (ISH) assay described here can detect RNA target sequences as short as 50 nucleotides at single-nucleotide resolution and at the single-cell level. This assay is complementary to the previously developed commercial assay and enables sensitive and specific in situ detection of splice variants, short targets, and point mutations within the tissue. In this protocol, probes were designed to target unique exon junctions for two clinically important splice variants, EGFRvIII and METΔ14. The detection of short target sequences was demonstrated by the specific detection of CDR3 sequences of T-cell receptors α and β in the Jurkat T-cell line. Also shown is the utility of this ISH assay for the distinction of RNA target sequences at single-nucleotide resolution (point mutations) through the visualization of EGFR L858R and KRAS G12A single-nucleotide variations in cell lines using automated staining platforms. In summary, the protocol shows a specialized RNA ISH assay that enables the detection of splice variants, short sequences, and mutations in situ for manual performance and on automated stainers.
FASEB J.
2017 Mar 03
Zhao J, Wu W, Zhang W, Lu YW, Tou E, Ye J, Gao P, Jourd'heuil D, Singer HA, Wu M, Long X.
PMID: 28258189 | DOI: 10.1096/fj.201601021R
Tetraspanins (TSPANs) comprise a large family of 4-transmembrane domain proteins. The importance of TSPANs in vascular smooth muscle cells (VSMCs) is unexplored. Given that TGF-β1 and myocardin (MYOCD) are potent activators for VSMC differentiation, we screened for TGF-β1 and MYOCD/serum response factor (SRF)-regulated TSPANs in VSMC by using RNA-seq analyses and RNA-arrays. TSPAN2 was found to be the only TSPAN family gene induced by TGF-β1 and MYOCD, and reduced by SRF deficiency in VSMCs. We also found that TSPAN2 is highly expressed in smooth muscle-enriched tissues and down-regulated in in vitro models of VSMC phenotypic modulation. TSPAN2 expression is attenuated in mouse carotid arteries after ligation injury and in failed human arteriovenous fistula samples after occlusion by dedifferentiated neointimal VSMC. In vitro functional studies showed that TSPAN2 suppresses VSMC proliferation and migration. Luciferase reporter and chromatin immunoprecipitation assays demonstrated that TSPAN2 is regulated by 2 parallel pathways, MYOCD/SRF and TGF-β1/SMAD, via distinct binding elements within the proximal promoter. Thus, we identified the first VSMC-enriched and MYOCD/SRF and TGF-β1/SMAD-dependent TSPAN family member, whose expression is intimately associated with VSMC differentiation and negatively correlated with vascular disease. Our results suggest that TSPAN2 may play important roles in vascular disease.-Zhao, J., Wu, W., Zhang, W., Lu, Y. W., Tou, E., Ye, J., Gao, P., Jourd'heuil, D., Singer, H. A., Wu, M., Long, X. Selective expression of TSPAN2 in vascular smooth muscle is independently regulated by TGF-β1/SMAD and myocardin/serum response factor.
Endocrinology.
2016 Oct 12
Burt PM, Xiao L, Dealy C, Fisher MC, Hurley MM.
PMID: 27732085 | DOI: 10.1210/en.2016-1548
Humans with X-linked hypophosphatemia (XLH) and Hyp mice, the murine homologue of the disease, develop severe osteoarthropathy and the precise factors that contribute to this joint degeneration remain largely unknown. Fibroblast growth factor 2 (FGF2) is a key regulatory growth factor in osteoarthritis. Although there are multiple FGF2 isoforms the potential involvement of specific FGF2 isoforms in joint degradation has not been investigated. Mice that overexpress the high molecular weight FGF2 isoforms in bone (HMWTg mice) phenocopy Hyp mice and XLH subjects and Hyp mice overexpress the HMWFGF2 isoforms in osteoblasts and osteocytes. Since Hyp mice and XLH subjects develop osteoarthropathies we examined whether HMWTg mice also develop knee joint degeneration at 2, 8, and 18-month-old compared with VectorTg (control) mice. HMWTg mice developed spontaneous osteoarthropathy as early as 2 months of age with thinning of subchondral bone, osteophyte formation, decreased articular cartilage thickness, abnormal mineralization within the joint, increased cartilage degradative enzymes, hypertrophic markers, and angiogenesis. FGF receptors 1 and 3 and fibroblast growth factor 23 were significantly altered compared to VectorTg mice. In addition, gene expression of growth factors and cytokines including bone morphogenetic proteins, Insulin like growth factor 1, Interleukin 1 beta, as well transcription factors Sex determining region Y box 9, hypoxia inducible factor 1 and nuclear factor kappa B subunit 1 were differentially modulated in HMWTg compared with VectorTg. This study demonstrates that overexpression of the HMW isoforms of FGF2 in bone results in catabolic activity in joint cartilage and bone that leads to osteoarthropathy.
Am J Pathol.
2017 Aug 17
Liu L, He Y, Guo K, Zhou L, Li X, Tseng M, Cai L, Lan ZJ, Zhou J, Wang H, Lei Z.
PMID: 28823874 | DOI: 10.1016/j.ajpath.2017.07.016
Gametogenetin binding protein 2 (GGNBP2) is an evolutionarily conserved zinc finger protein. Although Ggnbp2 null embryos in the B6 background died due to a defective placenta, 6.8% of Ggnbp2 null mice in the B6/129 mixed background were viable and continued to adulthood. Adult Ggnbp2 null males were sterile with smaller testes and an azoospermic phenotype, while mutant females were fertile. Histopathological analysis of 2-month-old Ggnbp2 null testes revealed absence of mature spermatozoa in the seminiferous tubules and epididymides and reduction of the number of spermatids. Ultrastructural analysis indicated dramatic morphological defects of developing spermatids in the Ggnbp2 null testes, including irregularly shaped acrosomes, acrosome detachment, cytoplasmic remnant, ectopic manchette and ill-formed head shape in both elongating and elongated spermatids. However, the numbers of spermatogonia, spermatocytes, Leydig cells and Sertoli cells in Ggnbp2 null testes did not significantly differ from the wild-type (WT) siblings. Gonadotropins, testosterone and the blood-testis barrier were essentially unaffected. Western blot analyses showed increases in α-E-catenin, β-catenin and N-cadherin, decreases in E-cadherin, afadin and nectin-3, and were unchanged in vinculin, nectin-2, FAK and integrin-β1 protein levels in Ggnbp2 null testes compared to WT siblings. Together, this study demonstrates that GGNBP2 is critically required for maintenance of the adhesion integrity of the adlumenal germ epithelium and is indispensable for normal spermatid transformation into mature spermatozoa in mice.
Cytometry A.
2017 Nov 30
Mavropoulos A, Allo B, He M, Park E, Majonis D, Ornatsky O.
PMID: 29194963 | DOI: 10.1002/cyto.a.23281
Mass cytometry uniquely enables high-dimensional single-cell analysis of complex populations. This recently developed technology is based on inductively coupled time-of-flight mass spectrometry for multiplex proteomic analysis of more than 40 markers per cell. The ability to characterize the transcriptome is critical for the understanding of disease pathophysiology, medical diagnostics, and drug discovery. Current techniques allowing the in situ detection of transcripts in single cells are limited to a small number of simultaneous targets and are generally tedious and labor-intensive. In this report, we present the development of a multiplex method for targeted RNA detection by combining the mass cytometry and RNAscope™ platforms. This novel assay, called Metal In Situ Hybridization (MISH), includes the hybridization of RNA-specific target probes followed by signal amplification achieved through a cascade of hybridization events, ending with the binding of amplifier-specific detector probes. The detector probes are tagged with isotopically pure metal atoms used for detection by mass cytometry. Proof-of-principle experiments show the simultaneous detection of three mRNA targets in Jurkat cells in suspension cell assay mode. The localization of transcripts was also investigated using the imaging mass cytometry platform in Jurkat and KG-1a cells. In addition, we optimized the antibody staining procedure to allow the co-detection of mRNA and cell surface markers. Our data demonstrate that MISH can be used to complement protein detection by mass cytometry as well as to investigate gene transcription and translation in single cells.
Peptides.
2017 Dec 28
Fernandez-Cachon ML, Pedersen SL, Rigbolt KT, Zhang C, Fabricius K, Hansen HH, Elster L, Fink LN, Schäfer M, Rhee NA, Langholz E, Wandall E, Friis SU, Vilmann P, Kristiansen VB, Schmidt C, Schreiter K, Breitschopf K, Hübschle T, Jorsal T, Vilsbøll T, Schm
PMID: 29289697 | DOI: 10.1016/j.peptides.2017.12.024
Abstract
AIM:
To determine whether intestinal expression of guanylate cyclase activator 2A (GUCA2A) and guanylate cyclase activator 2B (GUCA2B) genes is regulated in obese humans following Roux-en-Y gastric bypass (RYGB), and to evaluate the corresponding guanylin (GN) and uroguanylin (UGN) peptides for potentially contributing to the beneficial metabolic effects of RYGB.
METHODS:
Enteroendocrine cells were harvested peri- and post-RYGB, and GUCA2A/GUCA2B mRNA expression was compared. GN, UGN and their prohormones (proGN, proUGN) were administered subcutaneously in normal-weight mice to evaluate effects on food intake and glucose regulation. The effect of pro-UGN or UGN overexpression, using adeno-associated virus (AAV) vectors, was assessed in diet-induced obese (DIO) mice. Intracerebroventricular administration of GN and UGN was performed in rats for assessment of putative centrally mediated effects on food intake. GN and UGN, as well as their prohormones, were evaluated for effects on glucose-stimulated insulin secretion (GSIS) in rat pancreatic islets and perfused rat pancreas.
RESULTS:
GUCA2A and GUCA2B mRNA expression was significantly upregulated in enteroendocrine cells after RYGB. Peripheral administration of guanylins or prohormones did not influence food intake, oral glucose tolerance, and GSIS. Central administration of GN and UGN did not affect food intake in rats. Chronic AVV-mediated overexpression of UGN and proUGN had no effect on body weight or glucose homeostasis in DIO mice.
CONCLUSION:
GN and UGN, as well as their prohormones, do not seem to play a significant role in body weight regulation and glycemic control, suggesting that guanylin-family peptides do not show promise as targets for the treatment of obesity or diabetes.
Description | ||
---|---|---|
sense Example: Hs-LAG3-sense | Standard probes for RNA detection are in antisense. Sense probe is reverse complent to the corresponding antisense probe. | |
Intron# Example: Mm-Htt-intron2 | Probe targets the indicated intron in the target gene, commonly used for pre-mRNA detection | |
Pool/Pan Example: Hs-CD3-pool (Hs-CD3D, Hs-CD3E, Hs-CD3G) | A mixture of multiple probe sets targeting multiple genes or transcripts | |
No-XSp Example: Hs-PDGFB-No-XMm | Does not cross detect with the species (Sp) | |
XSp Example: Rn-Pde9a-XMm | designed to cross detect with the species (Sp) | |
O# Example: Mm-Islr-O1 | Alternative design targeting different regions of the same transcript or isoforms | |
CDS Example: Hs-SLC31A-CDS | Probe targets the protein-coding sequence only | |
EnEm | Probe targets exons n and m | |
En-Em | Probe targets region from exon n to exon m | |
Retired Nomenclature | ||
tvn Example: Hs-LEPR-tv1 | Designed to target transcript variant n | |
ORF Example: Hs-ACVRL1-ORF | Probe targets open reading frame | |
UTR Example: Hs-HTT-UTR-C3 | Probe targets the untranslated region (non-protein-coding region) only | |
5UTR Example: Hs-GNRHR-5UTR | Probe targets the 5' untranslated region only | |
3UTR Example: Rn-Npy1r-3UTR | Probe targets the 3' untranslated region only | |
Pan Example: Pool | A mixture of multiple probe sets targeting multiple genes or transcripts |
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