靶向小鼠CREPT基因CRISPR/Cas9敲除质粒的构建

目的 利用规律间隔成簇短回文重复序列/相关蛋白9(CRISPR/Cas9)系统构建靶向小鼠CREPT基因的敲除质粒。方法 根据CRISPR/Cas9靶点设计原则靶向小鼠CREPT基因的sgRNAs,与体外转录载体pUC57-sgRNA连接，构建CREPT基因敲除质粒；体外切割实验验证设计的sgRNAs是否具有活性。结果 设计了3条sgRNAs并分别准确插入pUC57-sgRNA载体；体外切割实验结果显示，以上3条sgRNAs均能够介导Cas9蛋白对靶片断进行切割。结论 制备了小鼠CREPT基因敲除质粒和体外转录的sgRNAs,为构建CREPT基因敲除小鼠模型奠定了重要基础。     

靶向c-myc基因的CRISPR/Cas9腺病毒系统对肝癌的抑制作用

目的:设计、构建并包装靶向c-myc基因的CRISPR/Cas9腺病毒;评估靶向c-myc基因的CRISPR/Cas9腺病毒系统对肝癌的抑制作用.方法:采用生物信息学网站设计靶向c-myc基因的gRNA,以GFP作为对照设计GFP gRNA;通过T7E1筛选出编辑效率高的gRNA并将筛选的gRNA构建CRISPR/Cas9腺病毒载体并包装腺病毒,通过分析细胞增殖、周期和凋亡及迁移能力的变化来评估靶向c-myc基因的CRISPR/Cas9腺病毒系统对肝癌的抑制作用.结果:成功设计、构建并包装靶向c-myc基因的CRISPR/Cas9腺病毒;靶向c-myc基因的CRISPR/Cas9腺病毒系统能够显著抑制Hepa1-6细胞的增殖和迁移、阻滞细胞周期进程,但对细胞凋亡无影响.结论:靶向c-myc基因的CRISPR/Cas9腺病毒系统可在细胞水平明显抑制肝癌细胞的生长.     

CRISPR/Cas9基因转录激活技术的建立及其应用

建立基于CRISPR/Cas9的基因转录激活技术,实现在HEK293细胞中激活cGAS基因的转录。首先利用慢病毒载体构建稳定表达Cas9的HEK293细胞;并采用Real-time PCR和western blot方法鉴定Cas9蛋白质表达效果。再选取不同靶序列,分别构建用于激活cGAS基因转录的sgRNA载体,将构建好的序列载体与CRISPR体系工具质粒共转染至稳定表达Cas9的HEK293细胞中。采用Real-time PCR方法检测细胞中内源cGAS的转录激活效果。成功建立了基于CRISPR/Cas9的基因转录激活技术,并在c GAS基因阴性表达的细胞中激活基因的转录。     

新型基因编辑技术CRISPR/Cas9系统研究现状

规律成簇间隔短回文重复序列及其相关系统(clustered regularly interspaced short palindromic repeats/CRISPR associated,CRISPR/Cas system)是细菌和古细菌防御外来噬菌体、质粒或其他外源DNA侵染的获得性免疫系统.依据Cas蛋白种类和同源性,CRISPR/Cas系统被分为3类,其中,Ⅰ类和Ⅲ类需要多种Cas蛋白参与,而Ⅱ类系统,即CRISPR/Cas9组成简单,仅需Cas9蛋白参与即可.经过遗传工程改造后的CRISPR/Cas9已经作为一种新型的基因编辑工具被用于多种生物的基因组编辑.本文就CRISPR/Cas9系统的发现、结构组成、作用机制、研究现状、面临的困境及应用前景等几方面进行了总结.     

基于CRISPR/Cas9技术构建TRPV1基因敲除的CACO-2稳定细胞系

基于CRISPR/Cas9基因编辑技术敲除人结直肠腺癌细胞Caco-2中的辣椒素受体基因TRPV1,构建TRPV1基因敲除的Caco-2稳定细胞系.使用CRISPR/Cas9在线靶点设计程序在TRPV1基因4个转录本的公共CDS区的第一个外显子DNA区域中设计一对gRNA,将gRNA构建到真核重组表达质粒的载体上,电转染待敲除细胞,经嘌呤毒素抗性筛选和基因测序获得敲除TRPV1基因的稳定细胞系.用Western Blot检测TRPV1基因蛋白表达量验证Caco-2中TRPV1基因的敲除效果,CCK-8检测TRPV1基因敲除细胞的生长曲线,与野生型细胞对比检测TRPV1基因敲除对细胞生长的影响.筛选出TRPV1基因敲除的CACO-2稳定细胞系,而且TRPV1基因敲除对Caco-2细胞生长无显著影响.基于CRISPR/Cas9基因编辑技术成功构建了TRPV1基因敲除的CACO-2稳定细胞系,为后续研究辣椒素对肠道脂类吸收影响的机理研究提供了有利的工具.     

CRISPR/Cas9技术联合AAV对Rosa-mTmG小鼠的MEF细胞进行编辑

目前,基因编辑技术已被广泛用于生物医学研究,本研究利用AAV(Adeno-Associated Virus)递送CRISPR/Cas9系统,实现高效率的基因编辑,并对特定组织细胞中的基因编辑进行了初步探索.我们首先采用Rosa-mTmG转基因小鼠来研究CRISPR/Cas9在小鼠胚胎成纤维细胞(Mouse Embryonic Fibroblast,MEF)中的编辑效率;接着,我们构建了能够被AAV包装的SaCas9系统,并通过瞬转以及AAV介导递送的方式检测其基因编辑效率;最后,我们还构建了含有心肌特异启动子的cTNT-PX601-sgRNA以备后续研究.结果发现,与瞬转相比,AAV介导CMV-PX601-sgRNA在体外能够极大地提高细胞的基因编辑效率.另外,cTNT-PX601-sgRNA能够在心肌细胞中特异表达.我们的结果表明,利用AAV介导的CRISPR/Cas9系统在体外可以实现高效地基因编辑,为靶向修复基因缺陷疾病,特别是特定组织器官中的基因缺陷,提供了研究工具和实验依据.     

CRISPR/Cas9核糖核蛋白介导的无T-DNA插入的白桦BpGLK1精准突变

【目的】CRISPR/Cas9 RNP系统是一种高效的基因编辑技术,具有简单、精准等特点,已被广泛应用于动、植物的基因编辑研究中。目前,通过微粒轰击技术将Cas9蛋白和gRNA核糖核蛋白复合体(RNP)导入受体细胞,获得无选择标记的基因编辑植物,该技术的提出为快速创制植物突变体提供了崭新思路。【方法】本研究以白桦(Betula platyphylla×B.pendula)BpGLK1基因为编辑的靶基因,采用CRISPR/Cas9 RNP技术开展无T-DNA插入的BpGLK1基因编辑。在白桦BpGLK1基因的第1外显子处设计靶位点,采用PCR扩增技术获得BpGLK1-E1靶位点片段;采用酶切及连接技术构建pAbAi-BpGLK1-E1重组质粒,将该质粒线性化后与Cas9蛋白作用,体外检测CRISPR/Cas9 RNP活性及gRNA的精准性。以白桦合子胚诱导的愈伤组织为受体,采用微粒轰击技术进行BpGLK1定向诱变。【结果】CRISPR/Cas9 RNP体外活性检测显示：Cas9蛋白及gRNA复合体具有裂解活性,可在BpGLK1靶位点的特定位点进行有效切割。以白桦成熟胚为材料,在无光照条件...     

ZFN,TALEN和CRISPR/Cas9在小鼠Rosa26基因定点整合外源基因的效率比较

哺乳动物黑色素的合成依赖于酪氨酸的氧化作用,而酪氨酸酶(Tyr)是催化酪氨酸氧化反应的关键酶,当外源Tyr基因整合进白毛色小鼠基因组中,会使它获得黑色素合成的能力,表现出与原来不同的毛色表型。为方便、快捷地获得Tyr基因整合的小鼠,构建了一个无启动子的pTyr-2A-DsRed同源重组质粒供体,选择Rosa26的第一个内含子作为外源基因整合的靶位点,设计了切割位点几乎一致的ZFN、TALEN和CRISPR/Cas9系统。通过流式对比分析C_2C_(12)细胞中红色荧光蛋白DsRed的表达水平,比较了3种基因组编辑工具介导的外源基因定点整合效率,结果发现CRISPR/Cas9的效率最高,在此基础上,利用CRISPR/Cas9将供体整合到小鼠胚胎干细胞中,筛选单细胞克隆进行囊胚腔注射和胚胎移植,获得一只存活的嵌合体小鼠,表现出白毛中夹杂黑毛的表型,表明整合到小鼠Rosa26的Tyr基因可以正常表达。     

CRISPR/dCas9在细菌转录调控中的应用及展望

CRISPR/dCas9是以CRISPR/Cas9系统为基础发展而来的转录调控工具.本文综述了CRISPR/dCas9作为转录抑制工具(CRISPRi)和转录激活工具(CRISPRa)的发展及应用,总结了在细菌中应用CRISPR/dCas9系统时转录调控系统的选择、构建、靶位点的选择以及gRNA的设计等方面存在的问题,并对相关解决方法进行了展望.     

利用CRISPR/Cas9敲除小鼠低温诱导蛋白(CIRBP)基因

为研究冷诱导RNA结合蛋白(CIRBP)与多种疾病的关系,利用CRISPR/Cas9系统敲除小鼠的CIRBP基因.针对CIRBP基因编码序列设计向导RNA(single guide RNA,sgRNA),构建相应sgRNA表达质粒.将此sgRNA与Cas9蛋白混合,注射C57BL/6小鼠的受精卵,实现靶基因敲除.通过T7E1酶切实验初步检测靶基因的修饰情况,再经过测序分析确定突变位点.获得了8个CIRBP基因突变的首建鼠,8只小鼠发生不同程度的碱基缺失.     

Markedness and its Interpretation in Language Use

Language markedness is a common phenomenon in languages, and is reflected from hearing, vision and sense, i.e. the variation in the three aspects such as phonology, morphology and semantics. This paper focuses on the interpretation of markedness in language use following the three perspectives, i.e. pragmatic interpretation, psychological interpretation and cognitive interpretation, with an aim to define the function of markedness.     

Lower Paleozoic oil relationships within Williston Basin, Canada

The Williston Basin is a significant petroleum province, containing oil production zones that include the Middle Cambrian to Lower Ordovician, Upper Ordovician, Middle Devonian, Upper Devonian and Mississippian and within the Jurassic and Cretaceous. The oils of the Williston Basin exhibit a wide range of geochemical characteristics defined as "oil families", although the geochemical signature of the Cambrian Deadwood Formation and Lower Ordovician Winnipeg reservoired oils does not match any "oil family". Despite their close stratigraphic proximity, it is evident that the oils of the Lower Palaeozoic within the Williston Basin are distinct. This suggests the presence of a new "oil family" within the Williston Basin. Diagnostic geochemical signatures occur in the gasoline range chromatograms, within saturate fraction gas chromatograms and biomarker fingerprints. However, some of the established criteria and cross-plots that are currently used to segregate oils into distinct genetic families within the basin do not always meet with success, particularly when applied to the Lower Palaeozoic oils of the Deadwood and Winnipeg Formation.     

An Analysis of Catherine＇s Character——Wuthering Heights

Wuthering Heights, Emily Bronte＇s only novel, was published in December of 1847 under the pseudonym Ellis Bell. The book did not gain immediate success, but it is now thought one of the finest novels in the English language. Catherine is the key character of this masterpiece, because everybody and everything center on her though she had a short life. We can understand this masterpiece better if we know Catherine well.     

Features of Women's language

Language is a means of verbal communication. People use language to communicate with each other. In the society, no two speakers are exactly alike in the way of speaking. Some differences are due to age, gender, statue and personality. Above all, gender is one of the obvious reasons. The writer of this paper tries to describe the features of women＇s language from these perspectives： pronunciation, intonation, diction, subjects, grammar and discourse. From the discussion of the features of women＇s language, more attention should be paid to language use in social context. What＇s more, the linguistic phenomena in a speaking community can be understood more thoroughly.     

Characterization of Ordovician carbonate reservoirs, southeastern Saskatchewan, Canada

The discovery of the prolific Ordovician Red River reservoirs in 1995 in southeastern Saskatchewan was the catalyst for extensive exploration activity which resulted in the discovery of more than 15 new Red River pools. The best yields of Red River production to date have been from dolomite reservoirs. Understanding the processes of dolomitization is, therefore, crucial for the prediction of the connectivity, spatial distribution and heterogeneity of dolomite reservoirs.The Red River reservoirs in the Midale area consist of 3～4 thin dolomitized zones, with a total thickness of about 20 m, which occur at the top of the Yeoman Formation. Two types of replacement dolomite were recognized in the Red River reservoir: dolomitized burrow infills and dolomitized host matrix. The spatial distribution of dolomite suggests that burrowing organisms played an important role in facilitating the fluid flow in the backfilled sediments. This resulted in penecontemporaneous dolomitization of burrow infills by normal seawater. The dolomite in the host matrix is interpreted as having occurred at shallow burial by evaporitic seawater during precipitation of Lake Almar anhydrite that immediately overlies the Yeoman Formation. However, the low δ18O values of dolomited burrow infills (-5.9‰～ -7.8‰, PDB) and matrix dolomites (-6.6‰～ -8.1‰, avg. -7.4‰ PDB) compared to the estimated values for the late Ordovician marine dolomite could be attributed to modification and alteration of dolomite at higher temperatures during deeper burial, which could also be responsible for its 87Sr/86Sr ratios (0.7084～0.7088) that are higher than suggested for the late Ordovician seawaters (0.7078～0.7080). The trace amounts of saddle dolomite cement in the Red River carbonates are probably related to "cannibalization" of earlier replacement dolomite during the chemical compaction.     

Design and analysis of generic LBS application developing platform

Location based services is promising due to its novel working style and contents.A software platform is proposed to provide application programs of typical location based services and support new applications developing efficiently. The analysis shows that this scheme is easy implemented, low cost and adapt to all kinds of mobile nework system.     

橡胶颗粒沥青混合料低温抗裂性试验研究

以AC-13级配为基础,将橡胶颗粒代替部分集料掺入混合料中,以低温弯曲试验为评价方法对不同橡胶颗粒掺量下沥青混合料的低温抗裂性进行研究,并引入应变能密度值对混合料的低温抗裂性进行综合评价.试验结果表明:橡胶颗粒沥青混合料试件的破坏微应变均超过2 300,满足冬寒区的技术指标;无论是否掺加橡胶颗粒,随着温度的下降,沥青混合料破坏时的最大弯拉强度增大,弯拉应变降低,劲度模量增大;弯曲应变能密度在胶粒掺量为1%左右时具有较大的弯曲应变能密度值,此时橡胶颗粒沥青混合料具有较好的低温抗裂性.     

低渗透非均质砂岩油藏启动压力梯度研究

理论推导与室内实验相结合,建立了低渗透非均质砂岩油藏启动压力梯度确定方法。首先借助油藏流场与电场相似的原理,推导了非均质砂岩油藏启动压力梯度计算公式。其次基于稳定流实验方法,建立了非均质砂岩油藏启动压力梯度测试方法。结果表明:低渗透非均质砂岩油藏的启动压力梯度确定遵循两个等效原则。平面非均质油藏的启动压力梯度等于各级渗透率段的启动压力梯度关于长度的加权平均;纵向非均质油藏的启动压力梯度等于各渗透率层的启动压力梯度关于渗透率与渗流面积乘积的加权平均。研究成果可用于有效指导低渗透非均质砂岩油藏的合理井距确定,促进该类油藏的高效开发。     

On Tragic Consciousness In The Works By Ernest Hemingway

As an American modern novelist who were famous in the literary world, Hemingway was not a person who always followed the trend but a sharp observer. At the same time, he was a tragedy maestro, he paid great attention on existence, fate and end-result. The dramatis personae's tragedy of his works was an extreme limit by all means tragedy on the meaning of fearless challenge that failed. The beauty of tragedy was not produced on the destruction of life, but now this kind of value was in the impact activity. They performed for the reader about the tragedy on challenging for the limit and the death.     

Quantitative trait loci （QTLs） for quality traits related to protein and starch in wheat

Quality traits in wheat （Triticum aestirum L.） were studied by quantitative trait locus （QTL） analysis in a recombinant inbred line （RIL） population, a set of 131 lines derived from Chuan 35050 × Shannong 483 cross （ChSh）. Grains from RILs were assayed for 21 quality traits related to protein and starch. A total of 35 putative QTLs for 19 traits with a single QTL explaining 7.99-40.52% of phenotypic variations were detected on 10 chromosomes, 1D, 2A, 2D, 3B, 3D, 5A, 6A, 6B, 6D, and 7B. The additive effects of 30 QTLs were positive, contributed by Chuan 35050, the remaining 5 QTLs were negative with the additive effect contributed by Shannong 483. For protein traits, 15 QTLs were obtained and most of them were located on chromosomes 1 D, 3B and 6D, while 20 QTLs for starch traits were detected and most of them were located on chromosomes 3D, 6B and 7B. Only 7 QTLs for protein and starch traits were co-located in three regions on chromosomes 1D, 2A and 2D. These protein and starch trait QTLs showed a distinct distribution pattern in certain regions and chromosomes. Twenty-two QTLs were clustered in 6 regions of 5 chromosomes. Two QTL clusters for protein traits were located on chromosomes 1D and 3B, respectively, three clusters for starch traits on chromosomes 3D, 6B and 7B, and one cluster including protein and starch traits on chromosome 1D.