scholarly journals Highly efficient neuronal gene knockout in vivo by CRISPR-Cas9 via neonatal intracerebroventricular injection of AAV in mice

Gene Therapy ◽  
2021 ◽  
Author(s):  
Sam Hana ◽  
Michael Peterson ◽  
Helen McLaughlin ◽  
Eric Marshall ◽  
Attila J. Fabian ◽  
...  
Keyword(s):  
Gene Knockout ◽  
Low Cost ◽  
High Specificity ◽  
Specific Gene ◽  
Preclinical Research ◽  
Guide Rnas ◽  
Neuronal Gene ◽  
Sgrna Design

AbstractCRISPR-Cas systems have emerged as a powerful tool to generate genetic models for studying normal and diseased central nervous system (CNS). Targeted gene disruption at specific loci has been demonstrated successfully in non-dividing neurons. Despite its simplicity, high specificity and low cost, the efficiency of CRISPR-mediated knockout in vivo can be substantially impacted by many parameters. Here, we used CRISPR-Cas9 to disrupt the neuronal-specific gene, NeuN, and optimized key parameters to achieve effective gene knockout broadly in the CNS in postnatal mice. Three cell lines and two primary neuron cultures were used to validate the disruption of NeuN by single-guide RNAs (sgRNA) harboring distinct spacers and scaffold sequences. This triage identified an optimal sgRNA design with the highest NeuN disruption in in vitro and in vivo systems. To enhance CRISPR efficiency, AAV-PHP.B, a vector with superior neuronal transduction, was used to deliver this sgRNA in Cas9 mice via neonatal intracerebroventricular (ICV) injection. This approach resulted in 99.4% biallelic indels rate in the transduced cells, leading to greater than 70% reduction of total NeuN proteins in the cortex, hippocampus and spinal cord. This work contributes to the optimization of CRISPR-mediated knockout and will be beneficial for fundamental and preclinical research.

scholarly journals Ursolic Acid Inhibits the Activation of Kupffer Cells by Caspase-11/NLRP3 Inflammasome Signaling Pathways

2021 ◽  
Author(s):  
Yuan Nie ◽  
Chen-kai Huang ◽  
Cong Liu ◽  
Xuan Zhu
Keyword(s):  
Liver Fibrosis ◽  
Ursolic Acid ◽  
Liver Damage ◽  
Kupffer Cells ◽  
Knockout Mice ◽  
Nlrp3 Inflammasome ◽  
Gene Knockout ◽  
Specific Gene

Abstract Background: Previous studies have indicated that Kupffer cells (KCs) are the main regulatory cells for the activation of hepatic stellate cells (HSCs), and caspase-11/NLRP3 inflammasome signaling plays crucial roles in the activation of monocyte-macrophages. Ursolic acid (UA) is a traditional Chinese medicine with antifibrotic effects, but the molecular mechanism underlying these effects is still unclear.Methods: A mouse primary Kupffer cell line in vitro and liver fibrosis mice (including specific gene knockout mice) in vivo were selected as experimental objects. RT-qPCR and Western blotting techniques were utilized to assess the mRNA and protein expression in each group. ELISA and histological analysis were utilized to assess liver injury and collagen deposition.Results: In vitro, caspase-11/NLRP3 inflammasome signaling promoted the activation of Kupffer cells, and UA inhibited the activation of Kupffer cells by caspase-11/NLRP3 inflammasome signaling. In vivo, UA reversed liver damage and fibrosis in fibrotic mice and was related to Kupffer cells; the expression of Caspase-11/NLRP3 inflammasome signaling in Kupffer cells of the UA group was inhibited. Even in the CCl4 group, the liver damage and fibrosis of NLRP3 knockout mice were alleviated, and related experiments also proved that the inhibitory effect of UA on Kupffer cells was related to the activation of the NLRP3 inflammasome.Conclusion: Caspase-11/NLRP3 inflammasome signal transduction is closely related to the activation of Kupffer cells and the occurrence of liver fibrosis. Additionally, caspase-11/NLRP3 inflammasome signaling serves as a new target for UA antifibrosis treatment.

26 THE USE OF PAIRED CRISPR GUIDE RNAs AND THE Cas9 SYSTEM DOES NOT ALWAYS PRODUCE SITE SPECIFIC DELETIONS OF GENE SEQUENCE IN PORCINE CELL AND EMBRYO CULTURE

2017 ◽  
Vol 29 (1) ◽  
pp. 120 ◽  
Author(s):  
A. M. Spate ◽  
K. M. Whitworth ◽  
C. W. O'Gorman ◽  
A. K. Byrne ◽  
R. S. Prather ◽  
...  
Keyword(s):  
Embryo Culture ◽  
Rna Structure ◽  
Low Cost ◽  
High Specificity ◽  
Promoter Sequence ◽  
Ease Of Use ◽  
T7 Promoter ◽  
Guide Rna ◽  
Guide Rnas

The use of the CRISPR/Cas9 system has become increasingly popular for creating gene edits in both cell and embryo culture. High specificity and efficiency of editing as well as low cost and ease of use has helped to promote its use. We hypothesised that by using multiple CRISPR guides at one time, we could quickly create exact deletions spanning greater areas of sequence. A total of 5 candidate genes (A, B, C, D, E) were targeted for deletions ranging in size of 74 to 551 bp. All modifications were created through the co-injection of 2 CRISPR guide RNAs with Cas9 RNA into in vitro-produced presumptive porcine zygotes. The CRISPR guides were created using gBlocks containing the T7 promoter sequence, 18–24 bp of CRISPR guide RNA, and 85 bp of tracer RNA. The RNA structure of each guide was reviewed using RNA Folding Form as well as offsite cutting using NCBI Blast. CRISPR guide RNA pairs (20 ng μL−1) and Cas9 RNA (20 ng μL−1) were co-injected (1–3 ρl) into the cytoplasm of IVF produced porcine zygotes using the FemtoJet 4i injector. Following injections, the zygotes were cultured in vitro for 5–6 days, and viable blastocyst or morula were selected for embryo transfer into recipient gilts. Resulting pigs were assayed for expected modifications using PCR. Pigs were considered modified if an insertion or deletion was measured by gel electrophoresis and DNA sequencing. Only one pair of CRISPR guides was injected per zygote, resulting in an individual PCR assay for the gene of interest. In total, 42 live piglets were born, 24 of which were edited, yielding 57% modification. When expected modifications v. observed were analysed, only 4 of 24 pigs (16%) produced the predicted modification on at least one allele. Of the remaining 20 pigs, several showed more than one form of modification. Insertions of ranging from 1 to 400 bp were detected in 10 pigs, 9 pigs formed biallelic modifications, 6 pigs produced altered sequence for greater than 2 alleles (mosaic), 6 pigs had deletions larger than the expected ranging from 11 to 1739 bp, and 14 had deletions smaller than the expected. Due to the absence of plasmid during injections, the insertions observed contained repetitive elements from the gene being modified as well as random additional bases. Additionally, CRISPR pairs were used in cell culture of porcine fibroblast modifying gene F, where they produced 6 different deletions ranging from the expected 63b to 617 bp. We recognise that the cutting efficiency of each CRISPR guide was not measured, as our goal was to create the expected deletions from pairs of CRISPR guides. We acknowledge our hypothesis was incorrect, as this data indicates that the CRISPR/Cas9 system is a very useful tool for gene editing, however it can induce unexpected modifications when used in pairs, in cell and embryo culture. Study was supported by funding from Food for the 21st Century and NIH (U42OD011140).

scholarly journals Tendon Stem/Progenitor Cells and Their Interactions with Extracellular Matrix and Mechanical Loading

2019 ◽  
Vol 2019 ◽  
pp. 1-10 ◽  
Author(s):  
Chuanxin Zhang ◽  
Jun Zhu ◽  
Yiqin Zhou ◽  
Bhavani P. Thampatty ◽  
James H-C. Wang
Keyword(s):  
Stem Cells ◽  
Extracellular Matrix ◽  
Progenitor Cells ◽  
Mechanical Loading ◽  
Gene Knockout ◽  
Deep Understanding ◽  
Future Research ◽  
Specific Gene

Tendons are unique connective tissues in the sense that their biological properties are largely determined by their tendon-specific stem cells, extracellular matrix (ECM) surrounding the stem cells, mechanical loading conditions placed on the tendon, and the complex interactions among them. This review is aimed at providing an overview of recent advances in the identification and characterization of tendon stem/progenitor cells (TSPCs) and their interactions with ECM and mechanical loading. In addition, the effects of such interactions on the maintenance of tendon homeostasis and the initiation of tendon pathological conditions are discussed. Moreover, the challenges in further investigations of TSPC mechanobiology in vitro and in vivo are outlined. Finally, future research efforts are suggested, which include using specific gene knockout models and single-cell transcription profiling to enable a broad and deep understanding of the physiology and pathophysiology of tendons.

A Physiologic Regulator of Collagen-Induced Platelet Aggregation: Inhibition by Clq

1981 ◽  
Vol 45 (02) ◽  
pp. 110-115 ◽  
Author(s):  
György Csákó ◽  
Eva A Suba
Keyword(s):  
Platelet Aggregation ◽  
Human Platelet ◽  
Platelet Reactivity ◽  
High Specificity ◽  
Turbidimetric Method ◽  
Specific Manner ◽  
Aggregation Inhibition ◽  
Different Tissues

SummaryPlatelet aggregations were studied by a turbidimetric method in citrated human platelet-rich plasmas (PRP) in vitro. Human Clq inhibited the aggregations caused by collagens derived from different tissues and species. Clq was needed by weight in comparable quantities to collagen for neutralizing the aggregating effect. The dependence of the inhibitory reaction on the preincubation of platelets with Clq and the differences in the occurrence of aggregating substances in supernatants of PRP triggered with collagen in the presence or absence of Clq, confirmed that Clq exerts its effect by preventing fixation of collagen to platelets. In addition, the high specificity of the inhibitory action of Clq for collagen-induced platelet aggregation was demonstrated by results obtained for testing a variety of aggregating agents in combination with Clq and/or collagen.Since normal concentrations of Clq in the blood are in the range of inhibitory doses of Clq for collagen-induced platelet aggregations in vitro and upon activation of complement Clq is known to dissociate from Cl, it is proposed that Clq may participate in a highly specific manner in regulating platelet reactivity to collagen in vivo.

Epithelial Organotypic Cultures: A Viable Model to Address Mechanisms of Carcinogenesis by Epitheliotropic Viruses

2018 ◽  
Vol 18 (4) ◽  
pp. 246-255 ◽  
Author(s):  
Lara Termini ◽  
Enrique Boccardo
Keyword(s):  
Three Dimensional ◽  
Cell Types ◽  
Experimental Models ◽  
Biological Research ◽  
Specific Gene ◽  
Specific Cell ◽  
Organotypic Cultures ◽  
Skin Physiology

In vitro culture of primary or established cell lines is one of the leading techniques in many areas of basic biological research. The use of pure or highly enriched cultures of specific cell types obtained from different tissues and genetics backgrounds has greatly contributed to our current understanding of normal and pathological cellular processes. Cells in culture are easily propagated generating an almost endless source of material for experimentation. Besides, they can be manipulated to achieve gene silencing, gene overexpression and genome editing turning possible the dissection of specific gene functions and signaling pathways. However, monolayer and suspension cultures of cells do not reproduce the cell type diversity, cell-cell contacts, cell-matrix interactions and differentiation pathways typical of the three-dimensional environment of tissues and organs from where they were originated. Therefore, different experimental animal models have been developed and applied to address these and other complex issues in vivo. However, these systems are costly and time consuming. Most importantly the use of animals in scientific research poses moral and ethical concerns facing a steadily increasing opposition from different sectors of the society. Therefore, there is an urgent need for the development of alternative in vitro experimental models that accurately reproduce the events observed in vivo to reduce the use of animals. Organotypic cultures combine the flexibility of traditional culture systems with the possibility of culturing different cell types in a 3D environment that reproduces both the structure and the physiology of the parental organ. Here we present a summarized description of the use of epithelial organotypic for the study of skin physiology, human papillomavirus biology and associated tumorigenesis.

scholarly journals A novel BRD4 inhibitor suppresses osteoclastogenesis and ovariectomized osteoporosis by blocking RANKL-mediated MAPK and NF-κB pathways

2021 ◽  
Vol 12 (7) ◽  
Author(s):  
Ying Liu ◽  
Wenjie Liu ◽  
Ziqiang Yu ◽  
Yan Zhang ◽  
Yinghua Li ◽  
...  
Keyword(s):  
Bone Loss ◽  
Osteoporosis Treatment ◽  
Inhibitory Effect ◽  
Specific Gene ◽  
Actin Ring ◽  
Treatment Target ◽  
Significant Inhibitory Effect ◽  
Promising Treatment

AbstractBromodomain-containing protein 4 (BRD4) has emerged as a promising treatment target for bone-related disorders. (+)-JQ1, a thienotriazolodiazepine compound, has been shown to inhibit pro-osteoclastic activity in a BRD4-dependent approach and impede bone loss caused by ovariectomy (OVX) in vivo. However, clinical trials of (+)-JQ1 are limited because of its poor druggability. In this study, we synthesized a new (+)-JQ1 derivative differing in structure and chirality. One such derivative, (+)-ND, exhibited higher solubility and excellent inhibitory activity against BRD4 compared with its analogue (+)-JQ1. Interestingly, (-)-JQ1 and (-)-ND exhibited low anti-proliferative activity and had no significant inhibitory effect on RANKL-induced osteoclastogenesis as compared with (+)-JQ1 and (+)-ND, suggesting the importance of chirality in the biological activity of compounds. Among these compounds, (+)-ND displayed the most prominent inhibitory effect on RANKL-induced osteoclastogenesis. Moreover, (+)-ND could inhibit osteoclast-specific gene expression, F‐actin ring generation, and bone resorption in vitro and prevent bone loss in OVX mice. Collectively, these findings indicated that (+)-ND represses RANKL‐stimulated osteoclastogenesis and averts OVX-triggered osteoporosis by suppressing MAPK and NF-κB signalling cascades, suggesting that it may be a prospective candidate for osteoporosis treatment.

scholarly journals Proanthocyanidins against Oxidative Stress: From Molecular Mechanisms to Clinical Applications

2018 ◽  
Vol 2018 ◽  
pp. 1-11 ◽  
Author(s):  
Lingyu Yang ◽  
Dehai Xian ◽  
Xia Xiong ◽  
Rui Lai ◽  
Jing Song ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Signaling Pathways ◽  
Molecular Mechanisms ◽  
Metabolic Diseases ◽  
Low Cost ◽  
Natural Agents ◽  
Molecular Damage ◽  
And Control

Proanthocyanidins (PCs) are naturally occurring polyphenolic compounds abundant in many vegetables, plant skins (rind/bark), seeds, flowers, fruits, and nuts. Numerousin vitroandin vivostudies have demonstrated myriad effects potentially beneficial to human health, such as antioxidation, anti-inflammation, immunomodulation, DNA repair, and antitumor activity. Accumulation of prooxidants such as reactive oxygen species (ROS) exceeding cellular antioxidant capacity results in oxidative stress (OS), which can damage macromolecules (DNA, lipids, and proteins), organelles (membranes and mitochondria), and whole tissues. OS is implicated in the pathogenesis and exacerbation of many cardiovascular, neurodegenerative, dermatological, and metabolic diseases, both through direct molecular damage and secondary activation of stress-associated signaling pathways. PCs are promising natural agents to safely prevent acute damage and control chronic diseases at relatively low cost. In this review, we summarize the molecules and signaling pathways involved in OS and the corresponding therapeutic mechanisms of PCs.

Serial analysis of gene expression (SAGE) during porcine embryo development

2004 ◽  
Vol 16 (2) ◽  
pp. 87 ◽  
Author(s):  
Le Ann Blomberg ◽  
Kurt A. Zuelke
Keyword(s):  
Gene Expression ◽  
Functional Genomics ◽  
Embryo Development ◽  
Molecular Mechanisms ◽  
Physiological Role ◽  
Transgenic Animals ◽  
Specific Gene ◽  
Research Strategies

Functional genomics provides a powerful means for delving into the molecular mechanisms involved in pre-implantation development of porcine embryos. High rates of embryonic mortality (30%), following either natural mating or artificial insemination, emphasise the need to improve the efficiency of reproduction in the pig. The poor success rate of live offspring from in vitro-manipulated pig embryos also hampers efforts to generate transgenic animals for biotechnology applications. Previous analysis of differential gene expression has demonstrated stage-specific gene expression for in vivo-derived embryos and altered gene expression for in vitro-derived embryos. However, the methods used to date examine relatively few genes simultaneously and, thus, provide an incomplete glimpse of the physiological role of these genes during embryogenesis. The present review will focus on two aspects of applying functional genomics research strategies for analysing the expression of genes during elongation of pig embryos between gestational day (D) 11 and D12. First, we compare and contrast current methodologies that are being used for gene discovery and expression analysis during pig embryo development. Second, we establish a paradigm for applying serial analysis of gene expression as a functional genomics tool to obtain preliminary information essential for discovering the physiological mechanisms by which distinct embryonic phenotypes are derived.

scholarly journals Transcription of alpha-specific genes in Saccharomyces cerevisiae: DNA sequence requirements for activity of the coregulator alpha 1.

1993 ◽  
Vol 13 (11) ◽  
pp. 6866-6875 ◽  
Author(s):  
D C Hagen ◽  
L Bruhn ◽  
C A Westby ◽  
G F Sprague
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Binding Site ◽  
Dna Sequences ◽  
Point Mutations ◽  
Transcription Activation ◽  
Specific Gene ◽  
Binding Assays ◽  
Weak Binding

Transcription activation of alpha-specific genes in Saccharomyces cerevisiae is regulated by two proteins, MCM1 and alpha 1, which bind to DNA sequences, called P'Q elements, found upstream of alpha-specific genes. Neither MCM1 nor alpha 1 alone binds efficiently to P'Q elements. Together, however, they bind cooperatively in a manner that requires both the P' sequence, which is a weak binding site for MCM1, and the Q sequence, which has been postulated to be the binding site for alpha 1. We analyzed a collection of point mutations in the P'Q element of the STE3 gene to determine the importance of individual base pairs for alpha-specific gene transcription. Within the 10-bp conserved Q sequence, mutations at only three positions strongly affected transcription activation in vivo. These same mutations did not affect the weak binding to P'Q displayed by MCM1 alone. In vitro DNA binding assays showed a direct correlation between the ability of the mutant sequences to form ternary P'Q-MCM1-alpha 1 complexes and the degree to which transcription was activated in vivo. Thus, the ability of alpha 1 and MCM1 to bind cooperatively to P'Q elements is critical for activation of alpha-specific genes. In all natural alpha-specific genes the Q sequence is adjacent to the degenerate side of P'. To test the significance of this geometry, we created several novel juxtapositions of P, P', and Q sequences. When the Q sequence was opposite the degenerate side, the composite QP' element was inactive as a promoter element in vivo and unable to form stable ternary QP'-MCM1-alpha 1 complexes in vitro. We also found that addition of a Q sequence to a strong MCM1 binding site allows the addition of alpha 1 to the complex. This finding, together with the observation that Q-element point mutations affected ternary complex formation but not the weak binding of MCM1 alone, supports the idea that the Q sequence serves as a binding site for alpha 1.

scholarly journals Construction of adenovirus vectors simultaneously expressing four multiplex, double-nicking guide RNAs of CRISPR/Cas9 and in vivo genome editing

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Tomoko Nakanishi ◽  
Aya Maekawa ◽  
Mariko Suzuki ◽  
Hirotaka Tabata ◽  
Kumiko Sato ◽  
...  
Keyword(s):  
Target Gene ◽  
Animal Experiments ◽  
Adenovirus Vector ◽  
Mouse Embryonic Fibroblast ◽  
Newborn Mouse ◽  
Guide Rnas ◽  
One Step ◽  
Target Effect

AbstractSimultaneous expression of multiplex guide RNAs (gRNAs) is valuable for knockout of multiple genes and also for effective disruption of a gene by introducing multiple deletions. We developed a method of Tetraplex-guide Tandem for construction of cosmids containing four and eight multiplex gRNA-expressing units in one step utilizing lambda in vitro packaging. Using this method, we produced an adenovirus vector (AdV) containing four multiplex-gRNA units for two double-nicking sets. Unexpectedly, the AdV could stably be amplified to the scale sufficient for animal experiments with no detectable lack of the multiplex units. When the AdV containing gRNAs targeting the H2-Aa gene and an AdV expressing Cas9 nickase were mixed and doubly infected to mouse embryonic fibroblast cells, deletions were observed in more than 80% of the target gene even using double-nicking strategy. Indels were also detected in about 20% of the target gene at two sites in newborn mouse liver cells by intravenous injection. Interestingly, when one double-nicking site was disrupted, the other was simultaneously disrupted, implying that two genes in the same cell may simultaneously be disrupted in the AdV system. The AdVs expressing four multiplex gRNAs could offer simultaneous knockout of four genes or two genes by double-nicking cleavages with low off-target effect.

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