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Search results for: cDNA Library Human Adult Normal Tissue Brain

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#31740838   2019/11/18 To Up

In situ readout of DNA barcodes and single base edits facilitated by in vitro transcription.

Molecular barcoding technologies that uniquely identify single cells are hampered by limitations in barcode measurement. Readout by sequencing does not preserve the spatial organization of cells in tissues, whereas imaging methods preserve spatial structure but are less sensitive to barcode sequence. Here we introduce a system for image-based readout of short (20-base-pair) DNA barcodes. In this system, called Zombie, phage RNA polymerases transcribe engineered barcodes in fixed cells. The resulting RNA is subsequently detected by fluorescent in situ hybridization. Using competing match and mismatch probes, Zombie can accurately discriminate single-nucleotide differences in the barcodes. This method allows in situ readout of dense combinatorial barcode libraries and single-base mutations produced by CRISPR base editors without requiring barcode expression in live cells. Zombie functions across diverse contexts, including cell culture, chick embryos and adult mouse brain tissue. The ability to sensitively read out compact and diverse DNA barcodes by imaging will facilitate a broad range of barcoding and genomic recording strategies.
Amjad Askary, Luis Sanchez-Guardado, James M Linton, Duncan M Chadly, Mark W Budde, Long Cai, Carlos Lois, Michael B Elowitz

2614 related Products with: In situ readout of DNA barcodes and single base edits facilitated by in vitro transcription.

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#30363057   // To Up

[SIVA1 Regulates the Stability of Single-Stranded DNA-Binding Protein 3 Isoforms].

The assembly of LIM-homeodomain (LIM-HD) transcriptional complex plays important roles in early neuronal development. The stability of LIM-HD is controlled by single-strand binding protein 3 (SSBP3) via a cascade mechanism protecting it from proteasomal degradation. The expression level of SSBP3 has to be precisely regulated. Although a decrease of SSBP3 level is associated with several diseases, the mechanism of SSBP3 downregulation and whether SSBP3 itself is subject to proteasomal degradation remain largely unknown. Two strongly conserved transcripts of the SSBP3 gene, SSBP3a and SSBP3c, were cloned from a human brain cDNA library. By RT-PCR, we show that Ssbp3c is continuously expressed in both embryonic and adult mouse brain, whereas Ssbp3a is restricted to embryonic brain tissue. By co-IP and GST pulldown assays, we identified SIVA1 as a novel SSBP3-binding factor. In a ubiquitination assay, we show that SIVA1 enhances the ubiquitination of SSBP3 and regulates its abundance. Our findings reveal the proteasomal degradation of SSBP3 for the first time and provide a rationale for an SIVAl-SSBP3-dependent mechanism for the disassembly of LIM-HD multiprotein complexes.
Z Yin, K Zhang, X Peng, Z Jiang, W Yuan, Y Wang, Y Li, X Ye, Y Dong, Y Wan, B Ni, P Zhu, X Fan, X Wu, X Mo

1368 related Products with: [SIVA1 Regulates the Stability of Single-Stranded DNA-Binding Protein 3 Isoforms].

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#30232282   2018/09/20 To Up

A limb-girdle muscular dystrophy 2I model of muscular dystrophy identifies corrective drug compounds for dystroglycanopathies.

Zebrafish are a powerful tool for studying muscle function owing to their high numbers of offspring, low maintenance costs, evolutionarily conserved muscle functions, and the ability to rapidly take up small molecular compounds during early larval stages. Fukutin-related protein (FKRP) is a putative protein glycosyltransferase that functions in the Golgi apparatus to modify sugar chain molecules of newly translated proteins. Patients with mutations in the FKRP gene can have a wide spectrum of clinical symptoms with varying muscle, eye, and brain pathologies depending on the location of the mutation in the FKRP protein. Patients with a common L276I FKRP mutation have mild adult-onset muscle degeneration known as limb-girdle muscular dystrophy 2I (LGMD2I), whereas patients with more C-terminal pathogenic mutations develop the severe Walker-Warburg syndrome (WWS)/muscle-eye-brain (MEB) disease. We generated fkrp-mutant zebrafish that phenocopy WWS/MEB pathologies including severe muscle breakdowns, head malformations, and early lethality. We have also generated a milder LGMD2I-model zebrafish via overexpression of a heat shock-inducible human FKRP (L276I) transgene that shows milder muscle pathology. Screening of an FDA-approved drug compound library in the LGMD2I zebrafish revealed a strong propensity towards steroids, antibacterials, and calcium regulators in ameliorating FKRP-dependent pathologies. Together, these studies demonstrate the utility of the zebrafish to both study human-specific FKRP mutations and perform compound library screenings for corrective drug compounds to treat muscular dystrophies.
Peter R Serafini, Michael J Feyder, Rylie M Hightower, Daniela Garcia-Perez, Natássia M Vieira, Angela Lek, Devin E Gibbs, Omar Moukha-Chafiq, Corinne E Augelli-Szafran, Genri Kawahara, Jeffrey J Widrick, Louis M Kunkel, Matthew S Alexander

1585 related Products with: A limb-girdle muscular dystrophy 2I model of muscular dystrophy identifies corrective drug compounds for dystroglycanopathies.

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#27117222   2016/04/27 To Up

In Vivo Selection Yields AAV-B1 Capsid for Central Nervous System and Muscle Gene Therapy.

Adeno-associated viral (AAV) vectors have shown promise as a platform for gene therapy of neurological disorders. Achieving global gene delivery to the central nervous system (CNS) is key for development of effective therapies for many of these diseases. Here we report the isolation of a novel CNS tropic AAV capsid, AAV-B1, after a single round of in vivo selection from an AAV capsid library. Systemic injection of AAV-B1 vector in adult mice and cat resulted in widespread gene transfer throughout the CNS with transduction of multiple neuronal subpopulations. In addition, AAV-B1 transduces muscle, β-cells, pulmonary alveoli, and retinal vasculature at high efficiency. This vector is more efficient than AAV9 for gene delivery to mouse brain, spinal cord, muscle, pancreas, and lung. Together with reduced sensitivity to neutralization by antibodies in pooled human sera, the broad transduction profile of AAV-B1 represents an important improvement over AAV9 for CNS gene therapy.
Sourav R Choudhury, Zachary Fitzpatrick, Anne F Harris, Stacy A Maitland, Jennifer S Ferreira, Yuanfan Zhang, Shan Ma, Rohit B Sharma, Heather L Gray-Edwards, Jacob A Johnson, Aime K Johnson, Laura C Alonso, Claudio Punzo, Kathryn R Wagner, Casey A Maguire, Robert M Kotin, Douglas R Martin, Miguel Sena-Esteves

1474 related Products with: In Vivo Selection Yields AAV-B1 Capsid for Central Nervous System and Muscle Gene Therapy.

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#25824290   2015/03/31 To Up

A library of MiMICs allows tagging of genes and reversible, spatial and temporal knockdown of proteins in Drosophila.

Here, we document a collection of ∼7434 MiMIC (Minos Mediated Integration Cassette) insertions of which 2854 are inserted in coding introns. They allowed us to create a library of 400 GFP-tagged genes. We show that 72% of internally tagged proteins are functional, and that more than 90% can be imaged in unfixed tissues. Moreover, the tagged mRNAs can be knocked down by RNAi against GFP (iGFPi), and the tagged proteins can be efficiently knocked down by deGradFP technology. The phenotypes associated with RNA and protein knockdown typically correspond to severe loss of function or null mutant phenotypes. Finally, we demonstrate reversible, spatial, and temporal knockdown of tagged proteins in larvae and adult flies. This new strategy and collection of strains allows unprecedented in vivo manipulations in flies for many genes. These strategies will likely extend to vertebrates.
Sonal Nagarkar-Jaiswal, Pei-Tseng Lee, Megan E Campbell, Kuchuan Chen, Stephanie Anguiano-Zarate, Manuel Cantu Gutierrez, Theodore Busby, Wen-Wen Lin, Yuchun He, Karen L Schulze, Benjamin W Booth, Martha Evans-Holm, Koen J T Venken, Robert W Levis, Allan C Spradling, Roger A Hoskins, Hugo J Bellen

2139 related Products with: A library of MiMICs allows tagging of genes and reversible, spatial and temporal knockdown of proteins in Drosophila.

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#25627687   2015/01/27 To Up

Cardiomyocyte expression and cell-specific processing of procholecystokinin.

Heart muscle cells produce peptide hormones such as natriuretic peptides. Developing hearts also express the gene for the classic intestinal hormone cholecystokinin (CCK) in amounts similar to those in the intestine and brain. However, cardiac expression of peptides other than natriuretic peptides has only been suggested using transcriptional measures or methods, with the post-translational phase of gene expression unaddressed. In this study, we examined the cardiac expression of the CCK gene in adult mammals and its expression at the protein level. Using quantitative PCR, a library of sequence-specific pro-CCK assays, peptide purification, and mass spectrometry, we demonstrate that the mammalian heart expresses pro-CCK in amounts comparable to natriuretic prohormones and processes it to a unique, triple-sulfated, and N-terminally truncated product distinct from intestinal and cerebral CCK peptides. Isoprenaline rapidly stimulated cardiac CCK gene expression in vitro and in vivo, which suggests that the cardiac-specific truncated pro-CCK may have pathophysiological relevance as a new marker of heart failure. The suggestion is confirmed by measurement of plasma from heart failure patients.
Jens P Goetze, Anders H Johnsen, Caroline Kistorp, Finn Gustafsson, Camilla B Johnbeck, Jens F Rehfeld

2446 related Products with: Cardiomyocyte expression and cell-specific processing of procholecystokinin.

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#25358671   2014/10/30 To Up

Differential increases of specific FMR1 mRNA isoforms in premutation carriers.

Over 40% of male and ∼16% of female carriers of a premutation FMR1 allele (55-200 CGG repeats) will develop fragile X-associated tremor/ataxia syndrome, an adult onset neurodegenerative disorder, while about 20% of female carriers will develop fragile X-associated primary ovarian insufficiency. Marked elevation in FMR1 mRNA transcript levels has been observed with premutation alleles, and RNA toxicity due to increased mRNA levels is the leading molecular mechanism proposed for these disorders. However, although the FMR1 gene undergoes alternative splicing, it is unknown whether all or only some of the isoforms are overexpressed in premutation carriers and which isoforms may contribute to the premutation pathology.
Dalyir I Pretto, John S Eid, Carolyn M Yrigollen, Hiu-Tung Tang, Erick W Loomis, Chris Raske, Blythe Durbin-Johnson, Paul J Hagerman, Flora Tassone

1777 related Products with: Differential increases of specific FMR1 mRNA isoforms in premutation carriers.

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#24743871   2014/04/17 To Up

Low expression of insulin-like growth factor binding protein 7 associated with poor prognosis in human glioma.

To investigate insulin-like growth factor binding protein 7 (IGFBP7) mRNA levels in human glioma and normal brain tissue, and to determine their clinical significance.
Xiangyang Tian, Ling Zhang, Laiguang Sun, Yihong Xue, Shuang Xie

2018 related Products with: Low expression of insulin-like growth factor binding protein 7 associated with poor prognosis in human glioma.

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#24308561   2013/12/06 To Up

Serological identification of URGCP as a potential biomarker for glioma.

Glioblastoma multiforme (GBM) is one of the most frequent human brain tumor and causes dismal outcome. To identify tumor-associated antigens in GBM patients may find potential diagnostic markers and immunotherapeutic targets. In this study, we identified a gene termed URGCP using the serological identification of antigens by recombinant A2B5 positive glioma cDNA library. The gene product of URGCP is immunogenic in GBM after tested in allogenic patients serum screening.
Ling-Chao Chen, Hai-Yan Zhang, Zhi-Yong Qin, Yang Wang, Ying Mao, Yu Yao, Liang-Fu Zhou

2230 related Products with: Serological identification of URGCP as a potential biomarker for glioma.

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