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Efforts to leverage clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) for targeted genomic modifications in mammalian cells are limited by low efficiencies and heterogeneous outcomes. To aid method optimization, we developed an all-in-one reporter system, including a novel superfolder orange fluorescent protein (sfOrange), to simultaneously quantify gene disruption, site-specific integration (SSI), and random integration (RI). SSI strategies that utilize different donor plasmid formats and Cas9 nuclease variants were evaluated for targeting accuracy and efficiency in Chinese hamster ovary cells. Double-cut and double-nick donor formats significantly improved targeting accuracy by 2.3–8.3-fold and 19–22-fold, respectively, compared to standard circular donors. Notably, Cas9-mediated donor linearization was associated with increased RI events, whereas donor nicking minimized RI without sacrificing SSI efficiency and avoided low-fidelity outcomes. A screen of 10 molecules that modulate the major mammalian DNA repair pathways identified two inhibitors that further enhance targeting accuracy and efficiency to achieve SSI in 25% of transfected cells without selection. The optimized methods integrated transgene expression cassettes with 96% efficiency at a single locus and with 53%–55% efficiency at two loci simultaneously in selected clones. The CRISPR-based tools and methods developed here could inform the use of CRISPR/Cas9 in mammalian cell lines, accelerate mammalian cell line engineering, and support advanced recombinant protein production applications. 相似文献
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Summary The cellular structures of acid rain-irrigated needles of several provenances of Norway spruce (Picea abies L. Karst) seedlings were studied after winter experimental freezing. Frost injuries and recovery were characterized by visual damage scoring and classification of mesophyll cell alterations, also using histochemical methods for carbohydrate fluorescent staining. The treatment with-30° C during the late dormancy period was sufficient to cause significant injuries and intracellular degradation in the tissues of the green needles. The most affected seedlings in terms of visual injury scoring were found among those treated with clean water or at pH 3, while freezing injury, defined as an occlusion of phenolic substances in the central vacuole of the mesophyll cells, was most abundant in the needles from spruces irrigated either with clean water or at pH 4 or pH 3. Electron microscopy revealed the details of the injury, e. g. thinning out of the cytoplasm and chloroplast stroma, darkening of the chloroplasts and eventually swelling of the chloroplasts and protoplast. PAS and ConA reactions in the needle tissue revealed intense starch accumulation in the mesophyll and transfusion tissues as early as in March, with a tendency to increase, especially in the untreated needles during the recovery period. Plasma membrane disturbances were indicated by histochemical identification of callose deposits in the mesophyll cell walls, these being most abundant in the acid rain-treated needles. All these findings suggest that freezing at –30° C was more deleterious to the seedlings pretreated with acid or clean water than to those not given additional irrigation. 相似文献
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目的 本研究旨在探讨细胞外基质刚度变化对神经干细胞(neural stem cells,NSCs)分化的影响及其作用机制。方法 本研究基于成功构建脊髓损伤大鼠模型,并制备不同刚度(0.7 kPa、40 kPa)的聚丙烯酰胺凝胶基底,将大鼠原代NSCs于不同刚度基底上培养。压电型机械敏感离子通道组件1(piezo type mechanosensitive ion channel component 1,Piezo1)shRNA质粒转染NSCs细胞。免疫荧光染色检测神经元标志物双皮质醇(doublecortion,DCX)和星形胶质细胞标志物胶质纤维酸性蛋白(glial fibrillary acidic protein,GFAP)阳性细胞百分比。免疫组织化学及蛋白质免疫印迹(Western blot)法检测损伤组织及NSCs细胞中Piezo1蛋白的表达水平。结果 与0.7 kPa基质刚度组相比,40 kPa基质刚度组中DCX阳性细胞数增加,而GFAP阳性细胞数减少,Piezo1蛋白表达量上升。脊髓损伤大鼠损伤组织Piezo1蛋白表达显著高于空白对照(sham)组。40 kPa基质刚度条件下沉默Piezo1后,DCX阳性细胞数减少,而GFAP阳性细胞数增加,差异具有统计学意义(P<0.05)。机制研究发现,沉默Piezo1导致IV型胶原及纤连蛋白表达下降。重组纤连蛋白逆转了Piezo1 shRNA对NSCs分化的影响,即DCX阳性细胞数增加,而GFAP阳性细胞数减少。结论 综上可见,硬基底刚度通过促进Piezo1蛋白表达,上调IV型胶原及纤连蛋白表达,从而调控NSCs细胞分化。本研究为基于生物材料治疗脊髓损伤提供了新的视角。 相似文献
107.
钙对臭氧伤害小麦的防护作用 总被引:8,自引:0,他引:8
春小麦分别用0、1‰、3‰、5‰浓度的CaCl_2溶液浸种及培养,9天后,用0.3—0.7ppm浓度的臭氧(O_3)对幼苗熏气0、4、8、12h,观测到来经Ca~(2+)处理材料的叶片,其外渗液紫外吸收值和电导率随O_3处理时间延长都有不同程度的升高,而叶内可溶性糖和叶绿素含量都明显降低,经过3‰Ca~(2+)处理O_3熏气4—12h的材料,其外渗液紫外吸值、电导率、外渗糖、蒸腾强度都低于对照,其叶内可溶性糖及叶绿素含量均高于对照,证收明在一定O_3浓度范围内,3‰CaCl_2对O_3侵害小麦有明显的防护作用。 相似文献
108.
冷害条件下凤眼莲某些生理特性变化的研究 总被引:24,自引:0,他引:24
研究了凤眼莲在冷害条件下细胞膜透性、体内生理代谢过程中一些大分子物质的变化。①0℃条件下细胞内大量电解质外渗,质膜性明显增加。此时,植株外部已发生了严重的伤害症状,②低温处理后,叶片内可溶性糖和游离脯氨酸累积,可溶性蛋白质含量基本不变。③过氧化物酶活性在低温下减弱,其同工酶谱带增加1-4条。过氧化氢酶活性明显增加,其同工酶谱带增加1-2条。SOD活性有所降低,其同工酶谱带在0℃、36小时后增加了1 相似文献
109.
ObjectiveGastric carcinoma (GC) has received extensive attention due to its complex pathogenesis. Studies have shown that the expression of Trefoil factor 1 (TFF1) and Partner and localiser of BRCA2 (PALB2) genes promotes the occurrence of GC. Therefore, we investigated whether TFF1 and PALB2 gene polymorphisms are associated with GC risk in the Chinese Han population.MethodsA total of 509 GC cases and 505 controls were recruited, and single nucleotide polymorphisms (SNPs) of TFF1 and PALB2 in these subjects were genotyped. The association between each candidate polymorphism and GC risk was assessed by calculating odds ratios (ORs) and 95% confidence intervals (CIs). The visualization of gene-gene interactions and functional enrichment analysis were then performed using Cytoscape software and the R package “cluster profile”.ResultsThe TFF1 rs2156310 polymorphism significantly reduced the predisposition to GC in people under 60 years of age (AA vs. AG - GG, OR = 0.58, 95% CI = 0.35–0.97, p = 0.036). The gender-stratified analysis found that PALB2 rs513313 was significantly associated with the risk of GC in males (CT vs. TT, OR = 1.51, 95% CI = 1.06–2.15, p = 0.022). Besides, PALB2 rs249954 significantly reduced the susceptibility to GC in females (AA vs GG, OR = 0.42, 95% CI = 0.19–0.94, p = 0.034).ConclusionOur results revealed that TFF1 and PALB2 gene polymorphisms were correlated with the genetic susceptibility to GC, providing certain data support for researchers to further study the mechanism of GC. 相似文献
110.
摘要 目的:评价放散式体外冲击波(rESW)穴位治疗联合中药熏洗在老年膝骨关节炎(KOA)中的应用效果。方法:选入2021年2月~2022年8月我院收治的老年KOA患者76例,根据治疗方法不同分为对照组和观察组,各38例,两组均予以中药熏洗,观察组加用rESW穴位治疗。评价两组的治疗效果、膝关节功能、疼痛程度等指标,并进行统计比较。结果:与治疗前相比,两组治疗2周、治疗4周时的VAS评分明显下降(P<0.05),而观察组下降幅度更大,与对照组差异显著(P<0.05);两组治疗前Lysholm评分无明显差异(P>0.05),而观察组治疗2周、治疗4周时的Lysholm评分显著高于对照组(P<0.05);观察组治疗显效率和有效率均显著高于对照组(P<0.05);观察组治疗2周、4周时的血清IL-1β、TNF-α和MMP-13水平较对照组低(P<0.05)。结论:rESW穴位联合中药熏洗治疗老年KOA患者疗效显著,可缓解疼痛症状,改善膝关节功能,控制炎症反应,且操作简单,值得推荐。 相似文献