转基因猪染色体原位杂交外源生长激素基因定位

近些年来随着原位杂交技术的不断改进,该技术已广泛用于染色体的基因定位。非放射性标记探针的应用使基因定位变得更加简单易行,从而有可能对动物的转基因进行定位研究。本文首次采用胶体金标记药盒（Anti-digoxigenin-gold)和银加强试剂（Silver enhance-ment reagents)的非同位素原位杂交技术对转基因猪外基因进行了定位研究。如Fig.1所示：表达质粒pSMTPGH含有载体pUC19,羊启动子MT011和猪生长激素PGH基因。选5头带有pSMTPGH的转基因猪,分别制备含有染色体DNA的杂交膜。用BglII和Smai对pSMTPGH进行完全酶切,收集0．9kb片段作为探针,以dig-11-dUTP进行标记。探针与DNA杂交后,用光学显微镜检查。选择分散良好、显影银颗粒清楚的玻片进行摄影记录（Fig.2)。对染色体上的显影银颗粒进行统计分析,参照家猪的染色体标准带型,确定外源PGH基因整合位点。Fig.3为4104号转基因猪染色体上的银颗粒分布情况。对5头转基因猪外源PGH基因定位的结果见Table1。探针的合理设计是外源基因定位研究成功的关键。本实验所用探针必须地与外源PGH基因杂交,而不受内源PGH基因的影响。我们设计的探针符合这一要求。采用dig11－dUTP标记探针,抗体金显色,银加强试剂放大杂交信号,在光学显微镜下可以直接观察杂交位点处的显影银颗粒,但于对实验进行统计分析。估计数据表明：转基因猪的外源PGH基因随机整合在所有染色体上,但在13号染色体上的机率略高。     

近交系小鼠双色荧光正相杂交芯片技术体系的建立和优化

目的探讨采用单核苷酸多态性（SNP）检测方法-双色荧光正相杂交芯片技术对近交系小鼠遗传质量监测及相关影响因素。方法运用基于芯片的双色荧光正相杂交检测SNP技术,进行芯片杂交动力学研究,考察信号值（Cy3,Cy5）和ratio值（Cy5/Cy3）与PCR产物点样浓度、PCR产物长度和荧光标记探针长度之间的关系,研究PCR产物点样浓度、PCR产物长度和荧光标记探针长度对SNP分型的影响。结果采用正反标记实验后,Ratio值随着PCR产物点样浓度的增加呈稳定趋势;PCR双链产物长度对信号值影响比较大,点样时其长度不宜太长,最好不超过450 bp;随荧光标记探针长度的增加,基因分型能力明显下降,长度为15 bp最佳,长度超过20 bp时,已基本没有区分能力。结论PCR产物点样浓度、PCR产物长度和荧光标记探针长度是双色荧光正相杂交SNP分型系统的重要影响因素,采取适当的PCR产物点样浓度、PCR产物长度和荧光标记探针长度,并采用正反标记实验,可以取得稳定、准确的基因分型效果。为进一步进行近交系小鼠遗传质量监测的研究奠定基础。     

总RNA和mRNA来源的探针与cDNA芯片杂交的差异研究

提取BEP2D细胞的总RNA并按两种方式进行cDNA芯片探针的标记,一种是将100μg BEP2D细胞的总RNA利用逆转录法直接标记成荧光探针,另一种是先从100μg BEP2D细胞的总RNA中分离出mRNA,然后再标记成荧光探针。将两份标记好的探针同时与含有230个基因的cDNA芯片杂交。杂交后的芯片经Axon4100B扫描仪扫描,发现两种方式标记探针的一致性为93．04％,并且mRNA来源探针杂交后的荧光信号值较总RNA的弱。探讨了这两种方法标记探针在基因芯片表达谱研究中的差异性,目的是为利用这两种方法标记探针进行基因表达谱研究提供一些依据。     

荧光标记寡核苷酸探针及其应用

寡核苷酸探针的标记非常重要。近年来 ,用荧光染料对探针进行非放射性标记受到很大重视 ,并取得了迅速发展 ,广泛应用于核酸序列测定、基因检测以及疾病诊断等。以下就寡核苷酸探针的荧光标记及其应用作一简要综述。     

生物素掺入PCR产物的反向杂交技术分析HLA基因型

本文介绍了与常规生物素或荧光标记引物不同,以及与同位素掺入ＰＣＲ产物不同的反向杂交技术,研究了生物素最佳掺入条件,测出加尾探针联结到尼龙膜上所需紫外光的强度,比较了生物素５＇端标记引物与生物素掺入ＰＣＲ产物的显色灵敏度,并根据实验测得的最佳条件分析了３个标准细胞株ＨＬＡ－ＤＰＢ_１基因型。     

生物素掺入PCR产物的反向杂交技术分析HLA基因型

本文介绍了与常规生物素或荧光标记引物不同,以及与同位素掺入ＰＣＲ产物不同的反向杂交技术,研究了生物素最佳掺入条件,测出加尾探针联结到尼龙膜上所需紫外光的强度,比较了生物素５＇端标记引物与生物素掺入ＰＣＲ产物的显色灵敏度,并根据实验测得的最佳条件分析了３个标准细胞株ＨＬＡ－ＤＰＢ１基因型。     

高分辨率熔解曲线法的非标记探针基因分型技术

PCR反应中利用荧光检测技术对已知位点进行基因分型时常采用荧光标记的寡核苷酸做探针。近年来新兴起的高分辨率熔解曲线技术可以采用非标记的探针对已知位点的SNP(single nucleotide polymorphism)或突变进行基因分型研究。采用非标记探针法对已知位点的基因分型研究具有廉价、快速、简便等特点,因此被大量应用在和疾病、形状等相关的一些多肽位点的研究中。本文较详细地介绍该技术的基本原理和实验中的注意事项。     

用原位分子杂交技术定位牛生长激素基因于5号染色体

本工作利用放射性标记的ｂＧＨ基因（３．０ｋｂ）为探针,通过原位杂交定位牛生长激素基因于染色体５ｑ２２－２６内。该结果与以前的ｂＧＨ基因定位的结果不同,讨论了基因探针、基因定位方法等方面与定位准确性的关系。     

双色原位杂交显示海带配子体5SrDNA与45SrDNA的非连锁关系

不等鞭毛类(stramenopiles)许多物种的报道认为,5SrRNA基因与其45SrRNA基因存在连锁关系,但通过设计不同引物进行PCR反应的探索,在海带中没有获得连锁的数据。为了从细胞学角度给予直观的证据,本研究基于海带5SrRNA及45SrRNA基因序列特征分析的基础上,利用双色荧光原位杂交(FISH)的技术和原理,以海带5SrRNA与45SrRNA基因的DNA为模板分别合成带有不同生物素荧光标记的探针,然后杂交到海带配子体的染色体上。FISH图片显示,红色标记的18S-5.8S-25S rDNA探针与绿色标记的5SrDNA探针,在同一幅海带配子体的染色体图片上都只出现了一个杂交信号;从大到小排列的核型显示,45SrDNA及5SrDNA分别定位于海带配子体的第23号和第27号染色体上,直观而清晰地表明5SrDNA并没有与45SrDNA连锁。这是首次尝试性地利用双色荧光原位杂交技术探讨藻类rDNA的染色体定位报道,这个结论有助于将FISH技术应用在海带染色体核型分析等研究。     

GFP质控芯片的初步研究

目的：建立一种质量控制芯片来监测样品标记、杂交和检测过程中的失误。方法：针对GFP基因设计的4条60mer寡核苷酸探针和1条阳性对照探针polv（U）与流感寡核苷酸探针一起打印在DAKO玻片上,并构建了GFP基因的克隆载体和体外表达载体,将从这两种重组载体上获得的绿色荧光蛋白（Green Fluorescent Protein,GFP）基因的ILNA、DNA片段和人的全血样品中的DNA用限制性显示技术（Restriction Display technology,RD）扩增标记,将标记的样品和荧光标记的通用引物U分别与芯片杂交、检测,并对扫描的结果进行统计分析。结果：GFP探针与相应的样品杂交时出现阳性信号,阳性对照探针在所有的杂交中均出现阳性信号,而空白对照则未检测荧光信号。结论：建立的质控芯片具有较好的敏感性和特异性,可以用于基因芯片中的质量监控。     

Fluorescent probes for imaging endogenous β-actin mRNA in living cells using fluorescent protein-tagged pumilio

Subcellular localization and dynamics of mRNAs control various physiological functions in living cells. A novel technique for visualizing endogenous mRNAs in living cells is necessary for investigation of the spatiotemporal movement of mRNAs. A pumilio homology domain of human pumilio 1 (PUM-HD) is a useful RNA binding protein as a tool for mRNA recognition because the domain can be modified to bind a specific 8-base sequence of target mRNA. In this study, we designed PUM-HD to match the sequence of β-actin mRNA and developed an mRNA probe consisting of two PUM-HD mutants flanking full-length enhanced green fluorescent protein (EGFP). Fluorescence microscopy with the probe in living cells revealed that the probe was labeled precisely with the β-actin mRNA in cytosol. Fluorescent spots from the probe were colocalized with microtubules and moved directionally in living cells. The PUM-HD mutants conjugated with full-length EGFP can enable visualization of β-actin mRNA localization and dynamics in living cells.     

Chromosomal localization of single copy genes SRY and SOX3 by primed in situ labeling (PRINS)

Primed in situ labeling (PRINS) is a sensitive and specific technique that can be used for the localization of single copy genes and DNA segments that are too small to be detected by conventional FISH. With PRINS, we physically localized the SRY gene to Yp11.31p11.32 and the SOX3 gene to Xq26q27. Locus-specific oligonucleotide primers were annealed in situ and extended on chromosome preparations fixed on microscope slides, in the presence of dATP, dCTP, dGTP, dTTP, biotin-16-dUTP, Tris-HCl, KCl, MgCl2, BSA, and Taq DNA polymerase. Fluorescent signals were detected in metaphase spreads and interphase nuclei. Our method may prove valuable for use with single copy genes in general.     

A new application of in situ hybridization: detection of numerical and structural chromosome aberrations with a combination centromeric-telomeric DNA probe

Fluorescent in situ hybridization provides a fast method for detection of specific nucleic acid sequences. We have used high-resolution, single-color fluorescent in situ hybridization with a combination centromeric-telomeric DNA probe, specific for chromosome 1, to investigate the feasibility of simultaneous assessment of numerical and structural chromosome aberrations. The K562 leukemia cell line served as a model.     

The molecular cytology of gene expression: fluorescent RNA as both a stain and tracer in vivo

For more than 60 years, RNA has been detectable in fixed cells and tissues by relatively specific staining methods. More recently, it has become possible to study RNA in unfixed, live cells. This review article describes how the intracellular dynamics and localization of RNA in vivo can be studied by microinjection of fluorescent RNA into cells- an approach we have termed Fluorescent RNA Cytochemistry. Depending on the particular RNA species under investigation, Fluorescent RNA Cytochemistry can operate as a "stain" to reveal intracellular sites at which a given RNA resides, or as a "tracer" to allow movements of a dynamically translocating RNA to be followed in the living cell. Several examples of Fluorescent RNA Cytochemistry are presented, collectively illustrating the range of applicability this approach offers in the toolbox of gene expression, studied as in vivo cell biology.     

地高辛配基标记探针在流行性出血热尸检组织中原位分子杂交技术的应用和探讨

将地高辛配基(Dig)标记的探针应用于流行性出血热(EHF)尸检组织的石蜡切片中,进行原位分子杂交四例。方法要点:(1)采用Dig标记的探针及碱性磷酸酶系统、提高其敏感性;(2)为达到被检核酸的暴露彻底、准确,选择合适的消化酶;(3)选择适当的杂交温度和时间,防止了非特异性背景,并避免了组织脱片。结果表明此方法操作简便、安全、快速、敏感。     

The human cystatin C gene (CST3), mutated in hereditary cystatin C amyloid angiopathy,is located on chromosome 20

Summary Hereditary cystatin C amyloid angiopathy has recently been shown to be caused by a point mutation in the cystatin C gene. To determine the chromosomal localization of the gene, 20 human-rodent somatic cell hybrids and a fulllength cystatin C cDNA probe were used. Southern blot analysis of BamHI digested cell hybrid DNA revealed that the probe recognizes a 10.6 kb human specific fragment and that this fragment cosegregates with human chromosome 20. Therefore, the human cystatin C gene (CST3) was assigned to chromosome 20.     

基于农杆菌介导瞬时转化法的AtGLR1.4亚细胞定位分析

将拟南芥基因AtGLR1.4启动子驱动的AtGLR1.4基因与绿色荧光蛋白(GFP)基因融合后,利用根瘤农杆菌介导瞬时转化法(Fast Agro-mediated Seedling Transfomation,FAST)浸染拟南芥幼苗,对其进行亚细胞定位的研究。转基因植株通过激光共聚焦扫描显微镜的观察,发现GFP绿色荧光在叶片表皮细胞的细胞膜上特异表达,表明At-GLR 1.4蛋白定位于细胞质膜上,为其后续的功能研究提供了线索。     

Selective enrichment of a large size genomic DNA fragment by affinity capture: an approach for genome mapping.

A method to enrich large size DNA fragments obtained by digestion with rare cutting restriction endonucleases was developed and applied for the isolation of a 150 kb SfiI fragment containing the beta-globin gene cluster. The digested DNA is rendered single stranded at the ends by diffusing a strand specific exonuclease into an agarose plug containing DNA. The plug is melted and solution hybridization is then performed with a bridge RNA containing specific sequences from the end of a desired fragment linked to a common probe sequence. The common probe sequence is annealed to a biotinylated RNA and the resulting tripartite hybrid is retained onto a solid matrix containing avidin and specifically released by ribonuclease action. Enrichments of greater than 350 fold have been achieved consistently. Such directed purification of large DNA fragments without cloning can considerably expedite mapping and gene localization in a complex genome and facilitate the construction of sublibraries from defined regions of the genome.