应用荧光原位杂交技术检测人β^E珠蛋白基因小鼠染色体上的整合状态

为将荧光原位杂交技术应用于基因定位研究中,探讨一种能有效地检测转基因动物染色体上外源基因整合状态的实验方法,对小鼠腹腔注射秋水仙素后,取转基因小鼠骨髓制备中期染色体,将传统的FISH方法加以改进,检测外源基因在转基因小鼠染色体上的整合状态。检测结果表明,外源人β^E珠蛋白基因已稳定地整合于小鼠染色体上,FISH能直观地反映外源基因在转基因动物染色体上的整合状态,该方法可对转基因动物及基因转移研究中的外源基因整合反进行染色体定位检测。     

应用荧光原位杂交技术检测人βE珠蛋白基因在转基因小鼠染色体上的整合状态

为将荧光原位杂交技术应用于基因定位研究中,探讨一种能有效地检测转基因动物染色体上外源基因整合状态的实验方法,对小鼠腹腔注射秋水仙素后,取转基因小鼠骨髓制备中期染色体,将传统的FISH方法加以改进,检测外源基因在转基因小鼠染色体上的整合状态.检测结果表明,外源人βE珠蛋白基因已稳定地整合于小鼠染色体上.FISH能直观地反映外源基因在转基因动物染色体上的整合状态,该方法可对转基因动物及基因转移研究中的外源基因整合后进行染色体定位检测。 Abstract:To determine the integration site of human βE globin gene in the chromosomes of transgenic mice, transgenic mice carrying human βE globin gene were injected intraperitoneally with colchicines, then, bone marrow cells wereisolated and metaphase chromosomes were prepared, the traditional FISH method was improved to detect the integration site of humanβE globin gene in transgenic mice when combined with G-banding. Human t3E globin gene can bedetected in different position of different chromosomes in transgenic mice and FISH signals showed that two mice were heterozygous of human 13E globin gene and one was homozygous. Human t3E globin gene was integrated into thechromosomes of transgenic mice in a random pattern and the results demonstrated that FISH can be used to investigate the integration site of foreign genes in transgenic mice.     

应用荧光原位杂交技术检测人βE珠蛋白基因在转基因小鼠染色体上的整合状态

为将荧光原位杂交技术应用于基因定位研究中,探讨一种能有效地检测转基因动物染色体上外源基因整合状态的实验方法,对小鼠腹腔注射秋水仙素后,取转基因小鼠骨髓制备中期染色体,将传统的FISH方法加以改进,检测外源基因在转基因小鼠染色体上的整合状态.检测结果表明,外源人βE珠蛋白基因已稳定地整合于小鼠染色体上.FISH能直观地反映外源基因在转基因动物染色体上的整合状态,该方法可对转基因动物及基因转移研究中的外源基因整合后进行染色体定位检测。 Abstract:To determine the integration site of human βE globin gene in the chromosomes of transgenic mice, transgenic mice carrying human βE globin gene were injected intraperitoneally with colchicines, then, bone marrow cells wereisolated and metaphase chromosomes were prepared, the traditional FISH method was improved to detect the integration site of humanβE globin gene in transgenic mice when combined with G-banding. Human t3E globin gene can bedetected in different position of different chromosomes in transgenic mice and FISH signals showed that two mice were heterozygous of human 13E globin gene and one was homozygous. Human t3E globin gene was integrated into thechromosomes of transgenic mice in a random pattern and the results demonstrated that FISH can be used to investigate the integration site of foreign genes in transgenic mice.     

应用荧光原位杂交检测人凝血因子Ⅸ在转基因小鼠染色体上的整合

应用荧光原位杂交（FISH）技术检测两个转基因小鼠家系从F1到F4代的整合情况。阳性转基因小鼠98%~100%的中期分裂相,85%~94%的间期核出现杂交信号;阴性对照小鼠100%的中期分裂相、95%~96%的间期核未出现杂交信号。结果表明,该FISH实验条件能对转基因整合位点进行高效特异检测。本文分析的两家系转基因小鼠均为单位点整合, 但整合位点不同。各家系内F1到F4代的转基因小鼠均可检出整合染色体,且整合位点相同,表明外源基因稳定整合并遗传给后代。 Abstract:Fluorescence in situ hybridization （FISH） was used to detect the integration of hFⅨ on chromosomes of transgenic mice from F1 to F4 generation in two strains.For transgenic mice,98%~100% of metaphases and 85%~94% of interphases showed hybridization signal.For negative control mice,100% of metaphases and 95%~96% of interphases showed no hybridization signal.The results demonstrated that FISH developed to detect the integration sites of hFⅨ was high efficient and specific.The integration sites of the transgenic mice analyzed were both single but different between the two strains.The integration chromosomes can be found in the transgenic mice from F1 to F4 generation and the integration sites were the same as each of the strains,which indicated that the transgene was stably integrated and transmitted to offspring.     

荧光原位杂交技术在染色体结构研究中的应用

1969年 Gall和 Pardue首先用同位素标记的 RNA探针定位特定的靶 DNA序列 ,开创了原位杂交技术 ,但同位素探针的高背景限制了它对靶序列的精确定位 ,随后发展了一系列非同位素方法 ,最初有生物素探针 ,用荧光素标记亲合素 ,酶联反应或胶体金检测 ,其他包括乙酰氨基荧光素 ( AAF) ,汞 ,地高辛 ( DIG)和 2 -三硝基酚等。荧光原位杂交 ( fluorescence in situ hybridiza-tion FISH)技术就是指将 DNA探针用特殊修饰的核苷酸分子标记 ,通过原位杂交与靶染色体或 DNA上特定的序列结合的方法学 ,靶染色体通常固定在载玻片上 ,杂交结果用荧…     

应用荧光原位杂交技术检测人类APPSWE基因在转基因小鼠染色体上的定位及位置效应

应用荧光原位杂交技术研究了人类淀粉样前体蛋白基因瑞典型突变（APPSWE）在转基因小鼠首建、F1及F2代小鼠染色体上的整合及定位,结果在2只首建转基因小鼠中,分别观察80个分裂相,出现杂交信号的核型分别为34及36个,检出率为42.5%和45%;1只F1及1只F2代转基因小鼠中,分别观察100个分裂相,出现杂交信号的核型分别为33及30个,检出率为33%和30%。转基因分别整合在8号、1号、17号和2号染色体上,提示转基因APPSWE已稳定整合到转基因小鼠的染色体上,并通过生殖细胞遗传给予子代,证实转基因在小鼠染色上的整合可能是随机的多点整合,同时,对不同整合位点的转基因小鼠进行了表型研究,结果发现不同整合位点对表型具明显影响。     

应用荧光原位杂交检测EB病毒BNLF-1基因在转基因小鼠子代染色体上的整合及其定位

应用荧光原位杂交技术研究了EB病毒潜伏膜蛋白基因(BNLF-1)在转基因小鼠子二代染色体上的整合及其定位。结果在两只子二代转基因小鼠中,分别观察80个和60个分裂相,出现杂交信号的核型分别为27和18个,检出率为33.8％和30％。转基因分别整合在14号染色体和10号染色体上。提示转基因BNLF-1已稳定整合到转基因小鼠的染色体上,并通过生殖细胞遗传给子代;推测转基因原代鼠的转基因整合可能是随机的多位点整合。     

应用荧光原位杂交检测EB病毒BNLF—1基因在转基因小鼠子代染色体上 …

应用荧光原位杂交技术研究了ＥＢ病毒潜伏膜蛋白基因（ＢＮＬＦ－１）在转基因小鼠子二代染色体上的整合及其定位。结果在两只子二代转基因小鼠中,分别观察８０个和６０个分裂相,出现杂交信号的核型分别为２７和１８个,检出率为３３．８％和３０％。转基因分别整合在１４号染色体和１０号染色体上。提示转基因ＢＮＬＦ－１已稳定整合到转基因小鼠的染色体上,并通过生殖细胞遗传给子代;推测转基因原代鼠的转基因整合可能是随机的     

荧光原位杂交技术在植物多倍体起源与进化研究中的应用

多倍化是植物物种形成与多样化的重要原动力。研究植物特别是一些重要经济作物和园艺植物多倍体的起源与进化,不仅对于揭示多倍体形成过程中性状变异的分子机制具有重要意义,而且可为植物遗传资源的保护与利用提供理论和技术支持。作为连接基因组序列片段到染色体组的桥梁,荧光原位杂交技术长期被广泛用来研究多倍体形成与进化过程中相关特异基因或序列的表达定位、外源染色体检测和鉴定、基因组结构变异等科学问题。因此,在简单介绍荧光原位杂交技术发展历史和植物多倍体主要类型的基础上,主要总结了荧光原位杂交技术在植物多倍体起源与进化相关研究上的应用。     

荧光原位杂交技术在植物细胞遗传学和绘制基因图谱中的应用现状与展望

荧光原位杂交是在分子水平上检测外源染色质的一种有效方法。其探针主要有染色体重复序列、总基因组DNA、寡单拷贝序列和染色体涂色集中等,该技术在研究植物细胞遗传学、基因扩增、基因作图及植物进化和亲缘关系的鉴定上已广泛应用。简要概述了荧光原位杂交技术在植物细胞遗传学和绘制基因图谱中的应用现状与展望。     

Chromosomal localization of silkworm (Bombyx mori) sericin gene 1 and chymotrypsin inhibitor 13 using fluorescence in situ hybridization

The chromosomal locations of two single-copy genes, Ser-1 and CI-13, in silkworm (Bombyx mori) were detected at the molecular cytogenetics level by fluorescence in situ hybridization in the study. The results showed that Ser-1 is located near the distal end of the 11th linkage group, relatively at the 12.5±1.4 position in pachytene; and that CI-13 has been mapped near the distal end of the 2nd linkage group, relatively at the 8.2±1.2 position in pachytene. Furthermore, their location model map-FISH map on silkworm chromosome was drawn. The FISH technique and its application to silkworm are also discussed in this paper.     

Location of ribosomal genes in CHO cells; in situ hybridization with a non-isotopic DNA probe on G-banded chromosomes.

A novel in situ hybridization technique using sulfonated probes is described. This non-radioactive approach, which employs chemically modified DNA and immunocytochemical procedures, is compatible with pre-G-banding and allows a rapid localization of the hybridized sequences on chromosomal spreads with a high spatial resolution. Using this technique we have localised the Chinese hamster ribosomal genes in the telomeric region of ten chromosomes, and among them in the subtelomeric q region of the Z5 chromosome. These results are discussed, the genetic markers confirming and locating the origin of Z group chromosomes by rearrangements of Chinese hamster chromosomes.     

莲C_0t-1 DNA的荧光原位杂交分析

为分析中国莲C_0t-1 DNA在其中期染色体上的分布,从中国莲基因组DNA中分离出C_0t-1 DNA,将基因组和所分离的C_0t-1 DNA用生物素标记后作探针,对中国莲染色体进行原位杂交。杂交结果用耦联有荧光素Cy3的生物素抗体检测,发现在每对染色体上均显示出特定的荧光原位杂交带。同时分析了FISH和GISH信号分布的异同。基于C_0t-1 DNA荧光原位杂交带型及染色体型,构建了中国莲核型。     

Evaluation of fluorescence in situ hybridization to detect encapsulated Bacillus pumilus SAFR-032 spores released from poly(methylmethacrylate)

Bacillus pumilus SAFR‐032 spores originally isolated from the Jet Propulsion Laboratory spacecraft assembly facility clean room are extremely resistant to UV radiation, H₂O₂, desiccation, chemical disinfection and starvation compared to spores of other Bacillus species. The resistance of B. pumilus SAFR‐032 spores to standard industrial clean room sterilization practices is not only a major concern for medical, pharmaceutical and food industries, but also a threat to the extraterrestrial environment during search for life via spacecraft. The objective of the present study was to investigate the potential of Alexa‐FISH (fluorescence in situ hybridization with Alexa Fluor® 488 labeled oligonucleotide) method as a molecular diagnostic tool for enumeration of multiple sterilant‐resistant B. pumilus SAFR‐032 spores artificially encapsulated in, and released via organic solvent from, a model polymeric material: poly(methylmethacrylate) (Lucite, Plexiglas). Plexiglas is used extensively in various aerospace applications and in medical, pharmaceutical and food industries. Alexa‐FISH signals were not detected from spores via standard methods for vegetative bacterial cells. Optimization of a spore permeabilization protocol capitalizing on the synergistic action of proteinase‐K, lysozyme, mutanolysin and Triton X‐100 facilitated efficient spore detection by Alexa‐FISH microscopy. Neither of the Alexa‐probes tested gave rise to considerable levels of Lucite‐ or solvent‐associated background autofluorescence, demonstrating the immense potential of Alexa‐FISH for rapid quantification of encapsulated B. pumilus SAFR‐032 spores released from poly(methylmethacrylate).     

莲C0t-1 DNA的荧光原位杂交分析

为分析中国莲Cot-1DNA在其中期染色体上的分布,从中国莲基因组DNA中分离出Cot-1DNA,将基因组和所分离的Cot-1DNA用生物素标记后作探针,对中国莲染色体进行原位杂交。杂交结果用耦联有荧光素Cy3的生物素抗体检测,发现在每对染色体上均显示出特定的荧光原位杂交带。同时分析了FISH和GISH信号分布的异同。基于Cot-1DNA荧光原位杂交带型及染色体型,构建了中国莲核型。     

Detection of Helicobacter pylori and determination of clarithromycin susceptibility using formalin-fixed, paraffin-embedded gastric biopsy specimens by fluorescence in situ hybridization

BACKGROUND: Clarithromycin resistance and poor compliance to therapy are often responsible for Helicobacter pylori eradication therapy failure. AIM: To evaluate fluorescence in situ hybridization (FISH) as a nonculture method to simultaneously detect H. pylori and to identify clarithromycin resistance. METHODS: Fifty-four patients with dyspepsia (17 male, 37 female subjects; mean age, 46.5; range, 21-78 years) were studied. Two antrum and corpus biopsies were taken from each patient. Positive rapid urease test (RUT) and histopathologic examinations defined H. pylori positivity. A total of 108 formalin-fixed paraffin-embedded gastric mucosal biopsies were examined retrospectively by the FISH (seaFAST H. pylori Combi-Kit) method. RESULTS: Forty-five patients (83.3%) were H. pylori positive and 43 (95.5%) were also positive by FISH. There were two false-positive FISH results. Fourteen patients (31.1%) had clarithromycin-susceptible strains, 4 (8.9%) resistant strains, and 27 (60%) both susceptible and resistant strains. CONCLUSION: FISH results correlated well with H. pylori infection and were able to identify clarithromycin-susceptible and -resistant strains. This technique will be helpful in determining the bacterial density and the success of treatment where clarithromycin has been widely used in populations to increase the efficacy of the treatment and to clarify the treatment failure in vitro.     

Chromosomal domains of chimpanzee are diverged from human as revealed by in situ hybridization using human genomic probe

Utilization of repetitive DNA probes to assess the taxonomic affinity between related species has become the most powerful tool in evolutionary biology today. Consequently, tremendous strides have recently been made towards establishing the phylogenetic relationship of humans with chimpanzee. We employed human genomic proe (P5080 B.5) to identify the degree of divergence of chimpanzee genome from humans. A small protion of structurally distinct genomic areas in chimpanzee could be identified by fluorescencein situ hybridization (FISH) technique when compared to human DNA. The genomic divergence is confined mainly to the chromosomal ends in chimpanzee and may be an important phylogenetic characteristic in human evolution.     

Nitrifying bacterial communities in an aquaculture wastewater treatment system using fluorescence in situ hybridization (FISH), 16S rRNA gene cloning, and phylogenetic analysis

Aquaculture, especially shrimp farming, has played a major role in the growth of Thailand's economy in recent years, as well as in many South East Asian countries. However, the nutrient discharges from these activities have caused adverse impacts on the quality of the receiving waterways. In particular nitrogenous compounds, which may accumulate in aquaculture ponds, can be toxic to aquatic animals and cause environmental problems such as eutrophication. The mineralization process is well known, but certain aspects of the microbial ecology of nitrifiers, the microorganisms that convert ammonia to nitrate, are poorly understood. A previously reported enrichment of nitrifying bacteria (ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB)) from a shrimp farm inoculated in a sequencing batch reactor (SBR) was studied by molecular methods. The initial identification and partial quantification of the nitrifying bacteria (AOB and NOB) were carried out by fluorescence in situ hybridization (FISH) using previously published 16S rRNA-targeting oligonucleotide probes. The two dominant bacterial groups detected by FISH were from the Cytophaga-Flavobacterium-Bacteroides and Proteobacteria (beta subdivision) phyla. Published FISH probes for Nitrobacter and Nitrospira did not hybridize to any of the bacterial cells. Therefore it is likely that new communities of NOBs, differing from previously reported ones, exist in the enrichments. Molecular genetic techniques (cloning, sequencing, and phylogenetic analysis) targeting the 16S rRNA genes from the nitrifying enrichments were performed to identify putative AOBs and NOBs.     

苜蓿种质间染色体多态性的荧光原位杂交检测

利用染色体荧光原位杂交技术(FISH),将3种重复序列5S rDNA、45S rDNA和C0t-1 DNA用不同荧光物进行标记,对我国10个不同地理来源的苜蓿种质(Medicago sativa L.;2n=4X=32)进行了染色体多态性检测。结果表明,利用以上重复序列可以较好地将苜蓿32条染色体区分为16对特征不同的染色体,10份不同种质材料FISH带纹特征表现高度相似,比较不同种质间同源染色体重复序列杂交特征,揭示出种质群体内和群体间多态性染色体的存在,其中不同的同源染色体多态性表现不尽一致,1号染色体(随体染色体)多态性最高,10份材料中检出7个多态型,3、4、15号染色体保守性较强,在不同种质间表现为单态,其他染色体多态性居中。对在地理分布上自西向东的10个材料进行染色体多态性比较,结果显示分布于西藏、新疆以及分布在辽宁的材料部分染色体多态型显著区别于其他材料。