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BackgroundMyostatin (MSTN) negatively regulates muscle mass and is a potent regulator of energy metabolism. However, MSTN knockout have affect mitochondrial function. This research assessed the mitochondrial energy metabolism of Mstn −/+ KO cells, and wondered whether the mitochondria biogenesis are affected.ResultsIn this study, we successfully achieved Mstn knockout in skeletal muscle C2C12 cells using a CRISPR/Cas9 system and measured proliferation and differentiation using the Cell-Counting Kit-8 assay and qPCR, respectively. We found that MSTN dysfunction could promote proliferation and differentiation compared with the behaviour of wild-type cells. Moreover, Mstn KO induced an increase in KIF5B expression. The mitochondrial content was significantly increased in Mstn KO C2C12 cells, apparently associated with the increases in PGC-1α, Cox1, Cox2, ND1 and ND2 expression. However, no differences were observed in glucose consumption and lactate production. Interestingly, Mstn KO C2C12 cells showed an increase in IL6 and a decrease in TNF-1α levels.ConclusionThese findings indicate that MSTN regulates mitochondrial biogenesis and metabolism. This gene-editing cells provided favourable evidence for animal breeding and metabolic diseases.How to cite: Wang L, Ding Q, Ma S, et al. CRISPR/Cas9-mediated MSTN gene editing Induced Mitochondrial Alterations in C2C12 myoblast Cells. Electron J Biotechnol 2019;40. https://doi.org/10.1016/j.ejbt.2019.03.009 相似文献
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The clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 9 (Cas9) genome editing system is a powerful tool for targeted gene modifications in a wide range of species, including plants. Over the last few years, this system has revolutionized the way scientists perform genetic studies and crop breeding, due to its simplicity, flexibility, consistency and high efficiency. Considerable progress has been made in optimizing CRISPR/Cas9 systems in plants, particularly for targeted gene mutagenesis. However, there are still a number of important challenges ahead, including methods for the efficient delivery of CRISPR and other editing tools to most plants, and more effective strategies for sequence knock-ins and replacements. We provide our viewpoint on the goals, potential concerns and future challenges for the development and application of plant genome editing tools. 相似文献
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We highlight the importance of the mixed genetic approaches (classical and currents) to improve the social perception related to the GMOs acceptance. We pointed out that CRISPR/Cas9 events could carry DNA variability/rearrangements related to somaclonal variations or epigenetic changes that are independent from the editing per se. The transformation of single cells, followed by plant regeneration, is used to generate modified plants, transgenic or genome editing (CRISPR/Cas9). The incidence of undesirable somaclonal variations and/or epigenetic changes that might have occurred during in vitro multiplication and regeneration processes, must be carefully analyzed in replicates in field trials. One remarkable challenge is related to the time lapse that selects the modified elite genotypes, because these strategies may spend a variable amount of time before the results are commercialized, where in all the cases it should be take into account the genotype × environment interactions. Furthermore, this combination of techniques can create an encouraging bridge between the public opinion and the community of geneticists who are concerned with plant genetic improvement. In this context, either transgenesis or genomic editing strategies become complementary modern tools to facing the challenges of plant genetic improvement. Their applications will depend on case-by-case analysis, and when possible will necessary associate them to the schemes and bases of classic plant genetic improvement.How to cite: Arencibia A, D’Afonseca V, Chakravarthi M, et al. Learning from transgenics: Advanced gene editing technologies should also bridge the gap with traditional genetic selection. Electron J Biotechnol 2019;41. https://doi.org/10.1016/j.ejbt.2019.06.001 相似文献
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Clustered regularly interspaced short palindromic repeats (CRISPR) and accompanying CRISPR-associated (Cas) proteins provide RNA-guided adaptive immunity for prokaryotes to defend themselves against viruses. The CRISPR-Cas systems have attracted much attention in recent years for their power in aiding the development of genome editing tools. Based on the composition of the CRISPR RNA-effector complex, the CRISPR-Cas systems can be divided into two classes and six types. In this review, we summarize recent advances in the structural biology of the CRISPR-Cas-mediated genome editing tools, which helps us to understand the mechanism of how the guide RNAs assemble with diverse Cas proteins to cleave target nucleic acids. 相似文献
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Jinshan Zhang Zhenyu Zhou Jinjuan Bai Xiaoping Tao Ling Wang Hui Zhang Jian-Kang Zhu 《国家科学评论(英文版)》2020,7(1):102
The microRNA miR396 directly represses GROWTH-REGULATING FACTORs (OsGRFs) and has been implicated in regulating rice yield and in nitrogen assimilation. Overexpressing the miR396 targets OsGRF4 and OsGRF6 improves rice yield via increased grain size and panicle branching, respectively. Here, we used CRISPR/Cas9 to assess the function of miR396 genes in rice. Knockout of MIR396ef (MIR396e and MIR396f), but not other isoforms, enhanced both grain size and panicle branching, resulting in increased grain yield. Importantly, under nitrogen-deficient conditions, mir396ef mutants showed an even higher relative increase in grain yield as well as elevated above-ground biomass. Furthermore, we identified OsGRF8 as a new target of miR396, in addition to the known targets OsGRF4 and OsGRF6. Disruption of the miR396-targeting site in OsGRF8 was sufficient to both enlarge grain size and elongate panicles. Our results suggest that rice-seed and panicle development are regulated by miR396ef-GRF4/6/8-GIF1/2/3 modules and that miR396ef are promising targets of genome editing for breeding environmentally friendly rice varieties that require less nitrogen fertilization. 相似文献
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BackgroundThe increasing rate of breast cancer globally requires extraordinary efforts to discover new effective sources of chemotherapy with fewer side effects. Glutaminase-free l-asparaginase is a vital chemotherapeutic agent for various tumor malignancies. Microorganisms from extreme sources, such as marine bacteria, might have high l-asparaginase productivity and efficiency with exceptional antitumor action toward breast cancer cell lines.Resultsl-Asparaginase-producing bacteria, Bacillus velezensis isolated from marine sediments, were identified by 16S rRNA sequencing. l-Asparaginase production by immobilized cells was 61.04% higher than that by free cells fermentation. The significant productivity of enzyme occurred at 72 h, pH 6.5, 37°C, 100 rpm. Optimum carbon and nitrogen sources for enzyme production were glucose and NH4Cl, respectively. l-Asparaginase was free from glutaminase activity, which was crucial medically in terms of their severe side effects. The molecular weight of the purified enzyme is 39.7 KDa by SDS-PAGE analysis and was ideally active at pH 7.5 and 37°C. Notwithstanding, the highest stability of the enzyme was found at pH 8.5 and 70°C for 1 h. The enzyme kinetic parameters displayed Vmax at 41.49 μmol/mL/min and a Km of 3.6 × 10−5 M, which serve as a proof of the affinity to its substrate. The anticancer activity of the enzyme against breast adenocarcinoma cell lines demonstrated significant activity toward MDA-MB-231 cells when compared with MCF-7 cells with IC50 values of 12.6 ± 1.2 μg/mL and 17.3 ± 2.8 μg/mL, respectively.ConclusionThis study provides the first potential of glutaminase-free l-asparaginase production from the marine bacterium Bacillus velezensis as a prospect anticancer pharmaceutical agent for two different breast cancer cell lines.How to cite: Mostafa Y, Alrumman S, Alamri S, et al. Enhanced production of glutaminase-free L-asparaginase by marine Bacillus velezensis and cytotoxic activity against breast cancer cell lines. Electron J Biotechnol 2019;42. https://doi.org/10.1016/j.ejbt.2019.10.001. 相似文献
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BackgroundMany human genetic diseases arise from point mutations. These genetic diseases can theoretically be corrected through gene therapy. However, gene therapy in clinical application is still far from mature. Nearly half of the pathogenic single-nucleotide polymorphisms (SNPs) are caused by G:C>A:T or T:A>C:G base changes and the ideal approaches to correct these mutations are base editing. These CRISPR-Cas9-mediated base editing does not leave any footprint in genome and does not require donor DNA sequences for homologous recombination. These base editing methods have been successfully applied to cultured mammalian cells with high precision and efficiency, but BE4 has not been confirmed in mice. Animal models are important for dissecting pathogenic mechanism of human genetic diseases and testing of base correction efficacy in vivo. Cytidine base editor BE4 is a newly developed version of cytidine base editing system that converts cytidine (C) to uridine (U).ResultsIn this study, BE4 system was tested in cells to inactivate GFP gene and in mice to introduce single-base substitution that would lead to a stop codon in tyrosinase gene. High percentage albino coat-colored mice were obtained from black coat-colored donor zygotes after pronuclei microinjection. Sequencing results showed that expected base changes were obtained with high precision and efficiency (56.25%). There are no off-targeting events identified in predicted potential off-target sites.ConclusionsResults confirm BE4 system can work in vivo with high precision and efficacy, and has great potentials in clinic to repair human genetic mutations.How to cite: Adlat S, Hayel F, Yang P, et al. CRISPR-mediated base editing in mice using cytosine deaminase base editor 4. Electron J Biotechnol 2021;52. https://doi.org/10.1016/j.ejbt.2021.04.010 相似文献
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Dalya Sh. Al-owaidi Moaed E. Algazally Alaa Sadeq Alawaad 《Indian journal of clinical biochemistry : IJCB》2021,36(3):304
This case–control study is aimed to evaluate serum concentration of Cyclin-Dependent Kinase 6 (CDK6) and the genetic association between rs2237572 CDK6 gene and breast cancer (BC) in Iraq. To attain this goal, 80 patients with BC as cases and 80 healthy individuals as controls were included. Further, BC patients were sorted according to the molecular classification into four subtypes of Luminal A, Luminal B, Her2/neu enriched and TPN. Serum concentration of CDK6 enzyme, allelic and genotypic frequencies of rs2237572 CDK6, and the occurrence of BC phenotype and its subtypes in the studied population were investigated. ELISA technique was used to perform the biochemical testing, while the molecular analysis was achieved by real-time PCR, high resolution melting analysis, conventional PCR, as well as sequencing analysis. The results revealed no significant difference in serum concentration of CDK6 enzyme between patients and healthy controls (p > 0.05). Also, no significant differences were shown between BC patients subtypes (p > 0.05). The rs2237572 CDK6 genotypes were associated with the BC and affirmed that allele C was inherited as a recessive risk factor. Moreover, a highly significant difference between patients’ subtypes in the genotypic frequency of rs2237572 (p < 0.01) was noted. Furthermore, the association of rs2237572 genotypes and CDK6 serum concentration in BC patients showed a considered significant difference between C/C and T/T, C/C and T/C and the CDK6 level (p < 0.05). Nevertheless, T/T and T/C did not show any significant difference with the CDK6 level. Hence, it was concluded that the rs2237572 of CDK6 gene is significantly correlated with BC. 相似文献
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BackgroundGain-of-function of fibroblast growth factor receptor 3 (FGFR3) is involved in the pathogenesis of many tumors. More and more studies have focused on the potential usage of therapeutic single-chain Fv (ScFv) antibodies against FGFR3. RNA interference (RNAi) has been considered as a promising therapeutic method against cancer. A tool which can deliver small interference RNAs (siRNAs) into FGFR3 positive cancer cells is very promising for anti-tumor therapy.ResultsIn this study, a novel fusion protein R3P, which consists of FGFR3-ScFv and protamine, was generated in Escherichia coli by inclusion body expression strategy and Ni-NTA chromatography. Its yield reached 10 mg per liter of bacterial culture and its purity was shown to be higher than 95%. 1 μg of R3P could efficiently bind to about 2.5 pmol siRNAs and deliver siRNAs into FGFR3 positive RT112 and K562 cells. Annexin V staining results showed that R3P can deliver the amplified breast cancer 1 (AIB1) siRNAs to induce RT112 cell apoptosis.ConclusionThese results indicated that R3P was a promising carrier tool to deliver siRNAs into FGFR3 positive cancer cells and to exert anti-tumor effect. 相似文献
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Jin-Jing Li Xiang Lin Cheng Tang Ying-Qian Lu Xinde Hu Erwei Zuo He Li Wenqin Ying Yidi Sun Lu-Lu Lai Hai-Zhu Chen Xin-Xin Guo Qi-Jie Zhang Shuang Wu Changyang Zhou Xiaowen Shen Qifang Wang Min-Ting Lin Li-Xiang Ma Ning Wang Adrian R Krainer Linyu Shi Hui Yang Wan-Jin Chen 《国家科学评论(英文版)》2020,7(1):92
We here report a genome-editing strategy to correct spinal muscular atrophy (SMA). Rather than directly targeting the pathogenic exonic mutations, our strategy employed Cas9 and guide-sgRNA for the targeted disruption of intronic splicing-regulatory elements. We disrupted intronic splicing silencers (ISSs, including ISS-N1 and ISS + 100) of survival motor neuron (SMN) 2, a key modifier gene of SMA, to enhance exon 7 inclusion and full-length SMN expression in SMA iPSCs. Survival of splicing-corrected iPSC-derived motor neurons was rescued with SMN restoration. Furthermore, co-injection of Cas9 mRNA from Streptococcus pyogenes (SpCas9) or Cas9 from Staphylococcus aureus (SaCas9) alongside their corresponding sgRNAs targeting ISS-N1 into zygotes rescued 56% and 100% of severe SMA transgenic mice (Smn−/−, SMN2tg/−). The median survival of the resulting mice was extended to >400 days. Collectively, our study provides proof-of-principle for a new strategy to therapeutically intervene in SMA and other RNA-splicing-related diseases. 相似文献
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SONG Jianlan 《中国科学院院刊(英文版)》2014,(4):285-287
A team of researchers at the Shanghai Institutes for Biological Sciences （SIBS）, CAS reported Oct .19 online in Nature the successful identificationof a long sought-after type of stem cells, the multipotent mammary stem cells （MaSCs） in mouse mammary gland, through a surface marker specifically expressed by this type of stem cells. This discovery settles a raging debate on the existence of multipotent MaSCs that can differentiate into all types of mammary epithelial cells, and moreover provides an ideal target for new drugs against breast cancers, especially a subtype that responds to no existing targeted therapy. 相似文献
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In vitro sensitivity testing of tumor cells could rationalize and improve the choice of chemotherapy and hormone therapy. In this report, a microfluidic device made from poly(dimethylsiloxane) and glass was developed for an assay of drug induced cytotoxicity. We evaluated the apoptotic and proliferation-inhibitory effects of anticancer drugs mitomycin C (MMC) and tamoxifen (TAM) using MCF-7 breast cancer cells. MMC and TAM both induced apoptosis and inhibited proliferation of MCF-7 cells in a concentration-dependent manner. MMC caused the expression of antiapoptotic protein Bcl-2 a dose-dependent reduction in MCF-7 cells. The expression of Bcl-2 did not change significantly in MCF-7 cells treated by TAM. The results in the microfluidic device were correlated well with the data obtained from the parallel experiments carried out in the conventional culture plates. The developed microfluidic device could be a potential useful tool for high content screening and high throughput screening research. 相似文献
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陈先军 《中国科技期刊研究》2015,26(11):1156-1160
[目的]根据论文的特点,从信息质量理论中提炼出有助于提高编辑初审工作质量的有效方法。[方法]根据Strong D M等人提出的信息质量定义,演绎推理论文信息质量的定义;根据信息质量的PSP/IQ模型,结合论文的特点和初审实践,从中归纳概括论文信息质量的维度,从而形成编辑初审的"论文信息质量5维法"。[结果]提出了学术期刊论文信息质量的概念,并探究了它的5个维度,形成了相应的论文信息质量5维法作为编辑初审的一种新方法。[结论]论文信息质量5维法在事实上和理论上都为编辑初审工作提供了一种可资借鉴的新思路和新方法。 相似文献
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Yu-Dong Zhang Suresh Chandra Satapathy David S. Guttery Juan Manuel Górriz Shui-Hua Wang 《Information processing & management》2021,58(2):102439
AimIn a pilot study to improve detection of malignant lesions in breast mammograms, we aimed to develop a new method called BDR-CNN-GCN, combining two advanced neural networks: (i) graph convolutional network (GCN); and (ii) convolutional neural network (CNN).MethodWe utilised a standard 8-layer CNN, then integrated two improvement techniques: (i) batch normalization (BN) and (ii) dropout (DO). Finally, we utilized rank-based stochastic pooling (RSP) to substitute the traditional max pooling. This resulted in BDR-CNN, which is a combination of CNN, BN, DO, and RSP. This BDR-CNN was hybridized with a two-layer GCN, and yielded our BDR-CNN-GCN model which was then utilized for analysis of breast mammograms as a 14-way data augmentation method.ResultsAs proof of concept, we ran our BDR-CNN-GCN algorithm 10 times on the breast mini-MIAS dataset (containing 322 mammographic images), achieving a sensitivity of 96.20±2.90%, a specificity of 96.00±2.31% and an accuracy of 96.10±1.60%.ConclusionOur BDR-CNN-GCN showed improved performance compared to five proposed neural network models and 15 state-of-the-art breast cancer detection approaches, proving to be an effective method for data augmentation and improved detection of malignant breast masses. 相似文献