外源DNA在哺乳动物细胞中不依赖于启动子的转录和剪接现象

外源性导入哺乳动物细胞的双链DNA会经历重组、重排及其他复杂的分子生化反应。极少数DNA分子会随机整合到宿主基因组,即发生水平基因转移。然而,目前还不清楚这些外源性DNA分子在不含有特殊启动子的情况下是否能被细胞的转录机器识别并起始转录。在构建环状RNA(circ RNA)表达体系的过程中,发现了外源性DNA分子能经历不依赖启动子的转录,而且转录本会发生符合"GU-AG"法则的RNA剪接进而产生类似于环状RNA的异常剪接分子。进一步研究表明,外源DNA在哺乳动物细胞中不依赖于启动子的转录和剪接现象是保守的。揭示了外源DNA在哺乳动物细胞中的一个新的命运途径,该发现对研究外源性DNA对宿主细胞的影响具有重要意义及应用价值。     

TLR9的特性及其介导的TLR9/CpG-DNA免疫信号通路

TLRs在天然免疫应答中发挥着重要作用,构成了机体抗感染的第一道防线,是沟通天然免疫和获得性免疫的桥梁。其中TLR9已经被证实能够识别细菌DNA中免疫刺激序列CpG,从而激活哺乳动物细胞的天然免疫机制,这一发现具有重要的生物学意义与应用价值。它不仅进一步推进了外源DNA激活哺乳动物抗感染天然免疫的进一步研究,更为CpG-DNA应用于抗感染、肿瘤和免疫缺陷疾病等的治疗提供新的思路,为改进DNA疫苗效果提供非常有益的帮助。     

昆虫杆状病毒应用于哺乳动物基因治疗的研究进展

杆状病毒是一类宿主特异性的昆虫病毒。昆虫杆状病毒表达系统是一个高效的真核表达系统,被广泛用于在昆虫细胞或昆虫幼虫中生产外源蛋白质。杆状病毒不能感染哺乳动物,却可以进入不同物种和组织来源的多种哺乳动物细胞,并在合适的哺乳动物启动子控制下表达外源基因。杆状病毒在哺乳动物细胞中不能复制,对细胞没有毒性,加上杆状病毒本身具有基因组大、可操作性好等优点,作为哺乳动物基因治疗的载体,将治疗基因传递给哺乳动物细胞已受到了广泛关注。在此就杆状病毒作为基因治疗载体的最新研究进展进行了阐述并探讨其发展趋势。     

杆状病毒介导外源基因在哺乳动物细胞中表达的研究进展

杆状病毒(Baculovirus)是一种以昆虫为唯一宿主的病毒, 可用做生物杀虫剂或作为表达载体在昆虫细胞中大量表达外源蛋白, 制备疫苗。研究发现, 在哺乳动物细胞中携带哺乳动物启动子的重组杆状病毒能启动下游外源基因的表达但病毒不能在哺乳动物细胞中增值, 对细胞毒性小, 转导成功的细胞可以稳定传代并有效表达外源基因, 哺乳动物细胞比昆虫细胞对蛋白质具有更好的翻译后修饰, 表达出的蛋白结构更接近天然蛋白。因此, 杆状病毒可作为一种新型的哺乳动物细胞基因转移载体, 用于表达外源基因及作为一种基因治疗载体, 具有巨大潜力, 日益受到人们的关注。本文对杆状病毒作为一种表达载体在哺乳动物细胞中表达的研究进展进行了综述。     

哺乳动物的基因工程(续二)

四、外源基因在哺乳动物细胞中的表达 本节所讨论的内容将涉及外源基因在哺乳动物受体细胞染色体上的整合作用以及加强外源基因表达的主要途径,即通过强启动子和增强子的作用或使外源基因在受体细胞中获得放大。     

用Ti质粒体外转化植物原生质体

进行遗传工程研究需要有一种方法把DNA引入宿主细胞,继而让其整合进宿主基因组并表达其基因功能。虽然,在哺乳动物、酵母和细菌中已有若干媒介DNA转移基因的方法。不过,还没有这样的方法可用于植物细胞。植物细胞吸收DNA的主要障碍是细胞壁,但用植物原生质体时,这一障碍是可所克服的。     

外源基因表达

体外重组DNA技术,在很多方面已经被用来导入外源遗传物质到一个新的细胞里,其目的常常是分离以及在结构上研究一个特定的DNA片段。可是,主要目的是在新的寄主细胞中获得外源基因的表达。 到达新寄主的某些外源基因,其表型至少在定性上与在原寄主中是一样的(表一)。     

用Ti质粒DNA离体转化植物原生质体成功

基因工程包括把DNA引入寄主细胞,并和寄主基因组整合,以及外源基因在寄主中表达。这在植物细胞里尚未被证明。最近新西兰的几位科学家首次在植物细胞里证明了这一点。他们是用烟草原生质体为实验材料,用Ti质粒DNA进行转化取得成功的。可引起冠缨瘤病的根瘤农杆菌是一个把外源DNA引入植物中去的天然的实验体系。这种根瘤农杆菌能把     

外源蛋白表达系统及利用植物表达外源蛋白的特点与优势

比较了大肠杆菌,酵母,昆虫细胞/杆状病毒,哺乳动物细胞,动物乳腺以及植物等不同受体作为外源蛋白表达系统的优缺点。论述了植物外源蛋白表达系统的特点,阐述利用植物生产外源蛋白的潜在优势。     

小鼠生殖系统中兔β-珠蛋白基因的引入

把克隆的外源基因引入组织培养的哺乳动物细胞的方法,已用来鉴定体内转录所必要的DNA序列。不过,这种系统对分析组织特异的基因表达所必要的序列的有用程度如何,现在仍不清楚。关于这种分析,更合适的途径也许是培养出一批这样的小鼠,即在它们的整个发育过程中,所有的细胞内均含有一种克隆的外源基因。这可以通过把一     

The Role of Cell Wall Revealed by the Visualization of Saccharomyces cerevisiae Transformation

Transformation is an indispensable method for the manipulation of Saccharomyces cerevisiae cell. The spf1 cell, in which the gene encoding an endoplasmic reticulum-located P-type ATPase is deleted, has been known to show the high-transformation phenotype. In this study, fluorescent microscopic observation of transformation process of S. cerevisiae using plasmid DNA labelled with fluorescent DNA probe, YOYO-1, suggested that the spf1 cell absorbed more plasmid DNA on cellular surface than did the wild-type cell and the unwashed cell did more plasmid DNA than the washed cell. The amounts of the absorbed DNA correlated with the transformation efficiency (number of transformants per μg plasmid DNA) and frequency (transformation efficiency per viable cell number). The high-transformation phenotype of spf1 cell and the effect of heat shock, which effectively induces the transformation of intact cell, disappeared upon cell wall digestion. Electron microscopic observation of the transformation process using negatively charged Nanogold as a mimic of plasmid DNA supported the result obtained using YOYO-1 and implied that plasmid DNA enters into cell together with membrane structure. These data strongly suggest that during the transformation of intact cell, plasmid DNA is initially absorbed on the cell wall, passes through the cell wall with the aid of heat shock, reaches to the membrane, and enters into the cell together with the membrane structure and that the capacity of the cell wall to absorb DNA is at least one of the determinants of transformation efficiency and frequency.     

准噶尔雅罗鱼β-肌动蛋白基因启动子的克隆及其活性功能初步检测

以开发利用新疆濒危鱼类准噶尔雅罗鱼(Leuciscus merzbacheri)基因资源为研究目的, 利用PCR技术克隆准噶尔雅罗鱼的β-actin 基因, 得到的β-actin 基因片段SZ21包含启动调控区, 大小为2 398 bp。SZ21的启动调控区包括β-actin 基因上游调控序列、第1、第2、第3和第4外显子部分序列。上游调控序列中含有对转录起重要作用的CAAT框、TATA 框和CArG 框等元件。对启动子序列在线分析表明, 获得的启动子含有E-box、RU49、ZBPF、CEBP、CREB等多个重要转录因子结合位点。用AatⅡ破坏真核表达载体pEGFP-N1-AFPⅢ中的CMV启动子, 将准噶尔雅罗鱼的SZ21启动调控区克隆到载体pEGFP-N1-AFPⅢ(CMV坏)上, 构建成重组表达载体β2 pEGFP-N1-AFPⅢ。脂质体转染BHK-21细胞。结果表明, 克隆的准噶尔雅罗鱼β-actin基因启动子SZ21具有启动EGFP报告基因在哺乳动物细胞中表达的活性。通过BHK-21绿色荧光细胞的传代证实, 克隆的启动子具有持续启动蛋白基因表达的活性, 在细胞传代中可以遗传。PCR检测传代的BHK-21绿色荧光细胞基因组DNA, 均能检测到SZ21目的片段。文章成功分离了具有活性功能的准噶尔雅罗鱼β-actin基因启动子。     

Instability of plasmid DNA integrated into mammalian somatic cell genome

The phenomenon of loosing exogenic DNA from the mammalian somatic cell genome is under investigation. It is found that foreign DNA incorporated into cell genome as a result of transfection by electrophoretion may be lost with the frequency from 1/100 up to 1/100 000 per cell division during cultivation. This effect is not dependent of the nature of cell line and vector DNA. It is actual for different cell lines: A23, human fibroblasts AG 11395, murine embryonic line F9, and for different plasmid vectors: p16, p.39, pATR4 and pcDNA3.1-Higr (WRN). Integration of pDNA into genome and the following loosing of this DNA is registered by selection markers G418 and hygromycin B resistance and gancyclovir sensibility. The presence of foreign DNA in the genome was controlled by PCR. It is found that true foreign DNA deletion from the genome takes place rather than gene expression changes. For closely linked plasmid genes deletion of both genes at once as well as loosing any one gene separately is shown. Thus, the phenomenon of selective deletion of exogenic DNA from genome has been demonstrated for different mammalian cells.     

精准高效外源DNA整合技术研究进展

精准高效整合技术是将外源DNA片段插入到目的细胞基因组特定位置的一项转基因技术。早期通过细胞基因组与外源DNA同源序列发生重组来完成靶向整合的目的。伴随基因编辑技术发展,特别是CRISPR/Cas9技术的出现,精准高效的外源DNA整合技术日益成熟,广泛应用到功能基因组学、转基因动物及遗传疾病治疗的研究中。围绕基因编辑研究进展、引导RNA修饰技术、单碱基整合技术、转座子技术和外源DNA整合效率等方面对精准靶向和高效整合技术进行综述。     

Reading Mammal Diversity from Flies: The Persistence Period of Amplifiable Mammal mtDNA in Blowfly Guts (Chrysomya megacephala) and a New DNA Mini-Barcode Target

Most tropical mammal species are threatened or data-deficient. Data collection is impeded by the traditional monitoring approaches which can be laborious, expensive and struggle to detect cryptic diversity. Monitoring approaches using mammal DNA derived from invertebrates are emerging as cost- and time-effective alternatives. As a step towards development of blowfly-derived DNA as an effective method for mammal monitoring in the biodiversity hotspot of Peninsular Malaysia, our objectives were (i) to determine the persistence period of amplifiable mammal mtDNA in blowfly guts through a laboratory feeding experiment (ii) to design and test primers that can selectively amplify mammal COI DNA mini-barcodes in the presence of high concentrations of blowfly DNA. The persistence period of amplifiable mammal mtDNA in blowfly guts was 24 h to 96 h post-feeding indicating the need for collecting flies within 24 h of capture to detect mammal mtDNA of sufficient quantity and quality. We designed a new primer combination for a COI DNA mini-barcode that did not amplify blowfly DNA and showed 89% amplification success for a dataset of mammals from Peninsular Malaysia. The short (205 bp) DNA mini-barcode could distinguish most mammal species (including separating dark taxa) and is of suitable length for high-throughput sequencing. Our new DNA mini-barcode target and a standardized trapping protocol with retrieval of blowflies every 24 h could point the way forward in the development of blowfly-derived DNA as an effective method for mammal monitoring.     

Replication, integration and stable germ-line transmission of foreign sequences injected into early zebrafish embryos

To generate stable lines of transgenic fish, early zebrafish embryos were injected with high concentrations of a linear bacterial plasmid. After injection, the foreign DNA was converted into a high molecular weight form and then amplified approximately tenfold during the initial rapid cleavages characteristic of the early embryo prior to gastrulation. While most of this DNA was subsequently degraded during gastrulation, some of the foreign sequences survived the gastrula stage and could be found in most of the injected fish at 3 weeks of age. Only about 5% of fish analysed 4 months after the injection retained foreign DNA in their fins, usually at less than one copy per cell. One of these fish was also found to contain about 100 copies per cell of foreign DNA in a fraction of its germ cells. Approximately 20% of the F1 offspring from this germ-line-positive parent inherited the foreign DNA, whereas 50% of F2 progeny obtained from an identified F1 individual inherited these sequences. The 50% transmission rate in F2 progeny was as expected for a single, heterozygous genomic insert. These observations indicate that injected DNA can be integrated into the fish genome, that the resulting transgenic fish are mosaic and that some of these mosaic individuals give rise to stable lines of transgenic fish.     

低压脉冲电泳介导的外源基因转化部分酶解的水稻小细胞团

开发了一类新型的低压脉冲电泳法介导外源基因进入水稻细胞的转化系统。本系统以水稻部分酶解小细胞团为受体,采用低压脉冲电泳推动质核DNA进入水稻细胞。以报告基因GUS酶活性为指标,借以测定转化了的水稻细胞。最佳的组合处理可以获得8.2％的转化频率。文中对低压脉冲电泳转移外源基因的条件亦作了讨论。