Synthesis of Digoxigenin-Labeled cRNA Probes for Nonisotopic in Situ Hybridization Using Reverse Transcription Polymerase Chain Reaction

Young, Iain D.; Stewart, R. J. C.; Ailles, Laurie; Mackie, Andrew S.; Gore, James

doi:10.3109/10520299309104687

Cited by 17 publications

(12 citation statements)

References 21 publications

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“…Sense and antisense riboprobes were synthesized using a PCR-based in situ hybridization technique as previously described (Suzuki et al, 2005;Young et al, 1993). Briefly, PCR was performed with TTLL3 gene-specific primers encompassing a T7 RNA polymerase-binding site.…”

Section: In Situ Hybridizationmentioning

confidence: 99%

Alterations in the balance of tubulin glycylation and glutamylation in photoreceptors leads to retinal degeneration

Grau

Masson

Gadadhar

et al. 2017

Journal of Cell Science

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Tubulin is subject to a wide variety of posttranslational modifications, which, as part of the tubulin code, are involved in the regulation of microtubule functions. Glycylation has so far predominantly been found in motile cilia and flagella, and absence of this modification leads to ciliary disassembly. Here, we demonstrate that the correct functioning of connecting cilia of photoreceptors, which are nonmotile sensory cilia, is also dependent on glycylation. In contrast to many other tissues, only one glycylase, TTLL3, is expressed in retina. Ttll3−/− mice lack glycylation in photoreceptors, which results in shortening of connecting cilia and slow retinal degeneration. Moreover, absence of glycylation results in increased levels of tubulin glutamylation in photoreceptors, and inversely, the hyperglutamylation observed in the Purkinje cell degeneration ( pcd) mouse abolishes glycylation. This suggests that both posttranslational modifications compete for modification sites, and that unbalancing the glutamylation-glycylation equilibrium on axonemes of connecting cilia, regardless of the enzymatic mechanism, invariably leads to retinal degeneration.

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Section: In Situ Hybridizationmentioning

confidence: 99%

Alterations in the balance of tubulin glycylation and glutamylation in photoreceptors leads to retinal degeneration

Grau

Masson

Gadadhar

et al. 2017

Journal of Cell Science

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“…Digoxigenin-labeled sense and antisense RNA probes for rat ET Areceptor mRNA were obtained by reverse transcription from the rat heart ET A and ET B -receptor cDNAs generated by PCR (see above) using T7-RNA polymerase and DIG-RNA-labeling mix (Boehringer, Mannheim, Germany) according to the method of Young (Young et al 1993).…”

Section: In Situ Hybridizationmentioning

confidence: 99%

Cellular localization of the endothelin receptor subtypes ET A and ET B in the rat heart and their differential expression in coronary arteries, veins, and capillaries

Wendel-Wellner¹,

Noll

König

et al. 2002

Histochemistry and Cell Biology

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In the heart, the endothelin (ET)/endothelin-receptor system is markedly involved in pathophysiological mechanisms underlying various cardiac diseases. Based upon pharmacological studies both ET-receptor subtypes take part in the regulation of coronary vascular tone, however, their detailed cellular distribution in the coronary vascular bed based upon direct mRNA and protein detection is unknown. This issue was addressed in the rat heart by means of non-radioactive in situ hybridization, RT-PCR, and immunohistochemistry. Expression of vascular ET(A)-receptors was detected in arterial smooth muscle and capillary endothelium while ET(B)-receptors were present in arterial, venous, and capillary endothelium, and in arterial and venous smooth muscle cells. This differential distribution of the ET-receptor subtypes supports the concept that ET(A)- as well as ET(B)-receptors mediate arterial vasoconstriction, while postcapillary vascular resistance is exclusively regulated by ET(B)-receptors. The observed capillary endothelial expression of the ET(A)-receptor correlates with the known ability of ET(A)-receptor antagonists to attenuate increases in cardiac microvascular permeability during endotoxin shock and ischemia/reperfusion injury.

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“…Specific probes were generated by PCR (21) so that sense primers were flanked by a SP6 promoter sequence and antisense primer was flanked by T7 promoter sequence. The RNA probes were labeled with the Dig RNA Labeling System (Roche Molecular Biochemicals), and hybridizations were carried out as previously described (22).…”

Section: Methodsmentioning

confidence: 99%

Impaired endochondral ossification and angiogenesis in mice deficient in membrane-type matrix metalloproteinase I

Zhou

Apte

Soininen

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

725

696

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Membrane-type matrix metalloproteinase I (MT1-MMP)-deficient mice were found to have severe defects in skeletal development and angiogenesis. The craniofacial, axial, and appendicular skeletons were severely affected, leading to a short and domed skull, marked deceleration of postnatal growth, and death by 3 wk of age. Shortening of bones is a consequence of decreased chondrocyte proliferation in the proliferative zone of the growth plates. Defective vascular invasion of cartilage leads to enlargement of hypertrophic zones of growth plates and delayed formation of secondary ossification centers in long bones. In an in vivo corneal angiogenesis assay, null mice did not have angiogenic response to implanted FGF-2, suggesting that the defect in angiogenesis is not restricted to cartilage alone. In tissues from null mice, activation of latent matrix metalloproteinase 2 was deficient, suggesting that MT1-MMP is essential for its activation in vivo. Matrix metalloproteinases (MMPs) are a family of Zndependent enzymes that are essential for extracellular matrix (ECM) turnover in normal and pathological conditions (1, 2). The MMPs are produced as latent proenzymes, and can be inhibited by specific tissue inhibitors of metalloproteinases (TIMPs). The enzymes can be divided into two structurally distinct subgroups, i.e., membrane-type (MT-MMP) and secreted MMPs. The secreted MMPs include interstitial collagenases (MMP-1, -8, and -13), which degrade fibrillar collagens, gelatinases (type IV collagenases, MMP-2 and -9) with high activity against gelatin and type IV collagen, and stromelysins (MMP-3, -10, and -11), which degrade a variety of collagenous and noncollagenous ECM proteins. Although the secreted MMPs have different expression patterns, there seems to be considerable redundancy and overlap between them with respect to function. Thus, mice deficient for MMP-2 (3), MMP-3 (4), MMP-7 (5), MMP-9 (6), MMP-10 (7), or MMP-12 (8) are all viable. Of these, only the MMP-9-deficient mice have been reported to show developmental abnormalities, which involve the growth plate and endochondral ossification (6).To date, five genetically distinct MT-MMPs (Mmp14-17, Mmp 21) have been identified (9-14). These enzymes (except MMP-17, which is glycosylphosphatidylinositol anchored) are singlepass type I membrane proteins with an extracellular N terminus containing the catalytic domain and a short C-terminal cytoplasmic domain. The prototypic MT-MMP, MT1-MMP (also termed MMP-14), was first identified as a cellular receptor and activator for pro-MMP-2 (9), but both MT1-MMP and MT2-MMP (MMP-15) have also been shown to have activity against a variety of ECM proteins, including gelatin, fibronectin, vitronectin, fibrillar collagens, and aggrecan (15, 16). MT1-MMP is widely expressed in cultured cells and tissues during development (17), but its strictly regulated spatial and temporal expression indicates more specific roles for this enzyme (17, 18). A crucial role for MT1-MMP for bone growth was recently demonstrated in MT1-MMP-deficient mi...

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Synthesis of Digoxigenin-Labeled cRNA Probes for Nonisotopic in Situ Hybridization Using Reverse Transcription Polymerase Chain Reaction

Cited by 17 publications

References 21 publications

Alterations in the balance of tubulin glycylation and glutamylation in photoreceptors leads to retinal degeneration

Alterations in the balance of tubulin glycylation and glutamylation in photoreceptors leads to retinal degeneration

Cellular localization of the endothelin receptor subtypes ET A and ET B in the rat heart and their differential expression in coronary arteries, veins, and capillaries

Impaired endochondral ossification and angiogenesis in mice deficient in membrane-type matrix metalloproteinase I

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