Duck hepatitis B virus DNA in liver, spleen, and pancreas: analysis by in situ and Southern blot hybridization

Tissues from a 10-week-old Pekin duck, experimentally infected at 1 day of age with duck hepatitis B virus (DHBV), were examined for the presence of replicative levels of DHBV DNA by in situ and Southern blot hybridization. Hepatocytes, pancreatic lymphoid follicle, exocrine, and endocrine cells, and splenic mononuclear cells all contained DHBV DNA localized predominantly to the cytoplasm of infected cells. Duck hepatitis B surface antigen distribution in the same tissues correlated well with the presence of DHBV DNA in many of these cells. In hepatocytes and pancreatic islet cells, 60% of DHBV DNA was present as single-stranded DNA, indicating the likelihood of ongoing virus replication in these cell types and providing further evidence that hepadnavirus DNA replication occurs largely within the cell cytoplasm. In contrast, DHBV in mononuclear cells within splenic germinal centers was wholly double-stranded, suggesting that limited, if any, DHBV DNA replication was occurring in this cell type. These data provide further information about the pathogenesis and cell-specific sites of DHBV infection.     

GEISHA, a whole-mount in situ hybridization gene expression screen in chicken embryos.

Despite the increasing quality and quantity of genomic sequence that is available to researchers, predicting gene function from sequence information remains a challenge. One method for obtaining rapid insight into potential functional roles of novel genes is through gene expression mapping. We have performed a high throughput whole-mount in situ hybridization (ISH) screen with chick embryos to identify novel, differentially expressed genes. Approximately 1,200 5' expressed sequence tags (ESTs) were generated from cDNA clones of a Hamburger and Hamilton (HH) stage 4-7 (late gastrula) chick embryo endoderm-mesoderm library. After screening to remove ubiquitously expressed cDNAs and internal clustering and after comparison to GenBank sequences, remaining cDNAs (representing both characterized and uncharacterized genes) were screened for expression in HH stage 3-14 embryos by automated high throughput ISH. Of 786 cDNAs for which ISH was successfully performed, approximately 30% showed ubiquitous expression, 40% were negative, and approximately 30% showed a restricted expression pattern. cDNAs were identified that showed restricted expression in every embryonic region, including the primitive streak, somites, developing cardiovascular system and neural tube/neural crest. A relational database was developed to hold all EST sequences, ISH images, and corresponding BLAST report information, and to enable browsing and querying of data. A user interface is freely accessible at http://geisha.biosci.arizona.edu. Results show that high throughput whole-mount ISH provides an effective approach for identifying novel genes that are differentially expressed in the developing chicken embryo.     

生物素标记探针原位杂交法检测细胞中c-myc基因的表达

本文采用生物素标记的c-mye基因作探针，通过细胞原位分子杂交技术，对人舌癌、膀胱癌和正常细胞中c-myc基因的转录量进行了定性和半定量分析。结果表明与正常细胞相比，舌癌和膀胱癌细胞中c-myc基因异常高度表达。     

A morphological analysis endocrine tumour genesis in pancreas and anterior pituitary of AVP/SV40 transgenic mice

Summary Insertion into the mouse genome of the hybrid oncogene made up of bovine vasopressin gene derived 5 upstream sequences and the coding sequences of SV40 large T-antigen promoted tumours in anterior pituitary and endocrine pancreas of mice bearing this transgene. In order to investigate the morphology of the steps in the neoplastic process, we used light and electron microscopy to study these organs in 42 animals belonging to the 3rd, 4th and 5th generations, subdivided into 4 age groups from 20 days to 100 days of life. Antibodies to large T-antigen were used to identify sites of expression of the hybrid oncogene, thus monitoring the steps in neoplastic transformation. Large T-antigen immunoreactivity was identified in dysplastic lesions of younger animals and in both dysplastic lesions and tumours of older mice. Insulin (100% of cases) and pancreatic polypeptide (25% of cases) immunoreactivities were revealed in pancreatic lesions but no hormonal immunoreactivity was detected in the pituitary lesions. The ultrastructural study confirmed that the majority cell population of the pancreatic neoplasms was B-type and that the anterior pituitary tumours were poorly granulated. The subcellular localization of large T-antigen immunoreactivity was investigated by the immunogold method and was confined to the heterochromatin of tumour cell nuclei. These findings provide evidence for the dysplasia-neoplasia sequence in the genesis of endocrine tumours of pituitary and pancreas of transgenic mice. The vasopressin-SV40 large T-antigen transgenic mice may therefore be an useful model for the study of endocrine cell oncogenesis,     

In situ hybridization study of pro-opiomelanocortin (POMC) gene expression in human pituitary corticotrophs and their adenomas

Summary Pro-opiomelanocortin (POMC) mRNA was detected on paraffin sections by in situ hybridization (ISH) in corticotrophs of 12 nontumorous pituitaries, 11 functioning corticotroph, and 11 silent pituitary adenomas. ISH combined with immunocytochemistry for adrenocorticotrophic hormone (ACTH), a POMC-derived peptide, was also performed. ACTH immunoreactive cells of the anterior lobes and those invading the posterior lobe showed a high or moderate level of POMC mRNA that was not correlated with the intensity of ACTH immunoreactivity. Variable levels of POMC gene expression were present in Crooke's cells, corticotrophs suppressed by glucocorticoid excess. Most functioning corticotroph adenomas and silent subtype 1 adenomas had an intense hybridization signal and ACTH immunoreactivity. In silent subtype 2 and 3 adenomas, POMC mRNA had a diffuse low level or was absent; in these adenomas ACTH immunoreactivity was diffuse, restricted to some cells, or negative. The results indicate that POMC gene is expressed in both normal and suppressed nontumorous corticotrophs. Intense signals for POMC mRNA are found in most functioning corticotroph adenomas. The difference between POMC gene expression in silent 1 and silent 2 and 3 adenomas suggests that different mechanisms are responsible for the lack of endocrine activity.     

Metal-inducible pathology in the liver, pancreas, and kidney of transgenic mice expressing SV40 early region genes.

Transgenic mice (SV-202) that carry the SV40 early region genes under the control of an inverted metallothionein promoter developed islet cell adenomas, hepatocellular carcinomas, and a generalized peripheral neuropathy. Both the islet cell adenomas and the hepatocellular carcinomas developed from the proliferation of T antigen-positive cells. However, T antigen expression was not seen in either the peripheral or central nervous systems. Stimulation of the metallothionein promoter with heavy metals altered the temporal onset of hepatic expression and broadened the distribution of oncogene expression to include exocrine pancreas and renal tubular epithelium. Although solid tumors were not seen in the exocrine pancreas or kidneys of SV-202 mice, all immunopositive tissues developed histologic changes. These results indicate that metallothionein-directed T antigen expression can induce abnormal cellular growth in a variety of tissues, and the distribution of these tissues can be manipulated with heavy metals.     

E-cadherin expression in colorectal cancer. An immunocytochemical and in situ hybridization study.

Expression of the epithelial-specific adhesion molecule E-cadherin has been assessed in paraffin-embedded tissue from a series of 72 colorectal carcinomas. Using immunocytochemistry and in situ hybridization it was found that E-cadherin expression was related inversely to tumor differentiation. Out of 44 well- and moderately differentiated tumors, 36 expressed good positivity, whereas 24 of 28 poorly differentiated tumors were E-cadherin-negative. Classification by Dukes stage revealed a highly significant difference (P << 0.001) between A and B (32 positive, four negative) and C1 and C2 (seven positive, 29 negative) stages in terms of immunoreactivity. Of the 32 lymph node metastases studied, 20 were negative for E-cadherin expression, as were seven of eight liver metastases. These results indicate that the down-regulation of E-cadherin levels in vivo is associated with the dedifferentiation, progression, and metastasis of colorectal cancer.     

Detection of N-myc gene expression in neuroblastoma tumors by in situ hybridization.

The presence of N-myc DNA amplification in human neuroblastoma tumors has been shown to be an independent prognostic factor predicting rapid progression of disease. Southern blot analysis has been used previously to detect N-myc amplification in these tumors. The authors report an analysis of N-myc gene expression by in situ hybridization in 28 human neuroblastoma tumors previously studied by Southern blot analysis. In the LA-N-5 human neuroblastoma cell line known to be amplified for N-myc, reaction conditions favoring RNA-RNA hybridization yielded an optimal signal. Using these hybridization conditions, in situ hybridization analysis of N-myc expression in 28 human neuroblastoma tissues correlated perfectly with N-myc DNA amplification in these tumors as detected by Southern blot analysis. In particular, there were no tumors in which N-myc RNA expression was found by in situ hybridization analysis in the absence of DNA amplification detectable by Southern blot, nor were there tumors that had DNA amplification in the absence of RNA expression. Heterogeneity of N-myc RNA expression was observed both among cells in any given tumor area, as well as within different areas of a single tumor. N-myc expression by in situ hybridization analysis was not observed in those tumors with more neuronally differentiated, ganglioneuroma histology. It is concluded that in situ hybridization of tissue sections is as effective as Southern blot analysis of tumor cell DNA in identifying human neuroblastoma tumors in which the N-myc gene is of prognostic significance.     

In situ hybridization in the study of pituitary tissues

This presentation summarizes the use of in situ hybridization in the study of pituitary abnormalities. The basic techniques, technical problems and pitfalls in in interpretation and the application of in situ hybridization are discussed.     

应用双色荧光原位杂交技术对转基因小鼠进行整合位点的染色体定位

目的 建立一种高度灵敏、特异的双色荧光原位杂交(dual-color fluorescence in situ hybridization,D-FISH)技术,对两种双阳性转基因小鼠外源基因进行整合位点的染色体定位.方法 对一只整合单纯疱疹病毒胸苷嘧啶激酶(herpes simplex virus thymidine kinase,HSV-tk)/增强型绿色荧光蛋白(enhanced green fluorescence protein,eGFP)的转基因小鼠以及两只整合RNA干扰载体(RNA interference,RNAi)的β654地中海贫血模型小鼠进行实验,脾脏细胞经培养后获得中期分裂相标本片,各加入适量生物素、地高辛标记探针混合液杂交,分别用罗丹明红色荧光抗体及FFIE绿色荧光抗体进行D-FISH检测.结果 两种转基因小鼠均能在同一个分裂相上同时检测到双色荧光信号.其中,HSV-tk/eGFP双阳性小鼠的分裂相上出现较强的绿色HSV-tk信号和红色eGFP信号,分别定位于染色体2E5-G3及8A2-A4;β654/RNAi双阳性小鼠检测到红色β654荧光信号及绿色RNAi荧光信号.经定位分析,β654均整合在染色体7D3-E2,RNAi病毒载体则是随机整合,其中一只鼠主要整合在1281位点,而另一只鼠主要整合在染色体1E2.3-1F、3A3两个位点.结论 用自行制备的DNA探针建立了高度灵敏、特异的D-FISH技术,同时结合G显带对双阳性转基因小鼠进行染色体基因定位.该技术平台的建立对于转基因动物和基因治疗动物模型的研究具有非常重要的意义.     

Identification, expression and in situ hybridization of an eggshell protein gene from Fasciola hepatica

The molecular basis of egg formation in the parasitic liver fluke, Fasciola hepatica, was investigated by isolating and characterizing an abundant cDNA from a female genital complex cDNA library. It was expressed in Escherichia coli as a beta-galactosidase fusion protein, which was purified and used to produce polyclonal antibodies. Using immunoblots, the antiserum recognized two soluble constituents of isolated egg shells, both significantly larger than predicted from cDNA sequencing. Using in situ hybridization, the message was detected in cells in the adult vitelline follicles. Eggshell protein mRNA expressed in E. coli will provide a source of precursor protein for further studies of parasite eggshell formation.     

Developmental changes in tropoelastin gene expression in the rat lung studied by in situ hybridization.

Gene expression for tropoelastin, the proprotein for elastin, was examined in the rat lung from 17 days of gestation (pseudoglandular stage) to adulthood by in situ hybridization using a rat-specific 35S-radiolabeled riboprobe. The tropoelastin message was present in vascular and airway smooth muscle, endothelial, septal interstitial, alveolar wall, and mesothelial cells but not in epithelial cells. With alveolar septal formation, the message in the interstitium increased progressively from 17 days of gestation, reaching a peak at 7 to 11 days postnatal. The signal in the arterial walls, in contrast, peaked between 19 days of gestation to 1 day postnatal and thereafter declined first from the outer media. The signal in general declined significantly by 21 days postnatal, and elastogenesis was virtually absent in the adult. These results support the idea that tropoelastin gene expression in the interstitium is closely associated with the centripetal progression of alveolarization, and the early postnatal decrease of tropoelastin expression in blood vessels corresponds with the sudden postnatal changes in the pulmonary hemodynamics. Furthermore, in the rat fetus and neonate, endothelial cells expressed the gene for tropoelastin and hence probably play a significant role in the formation of internal elastic lamina in vivo.     

Perforin gene expression in stimulated human peripheral blood T cells studied by in situ hybridization and northern blotting analysis.

In situ hybridization was used here to monitor the mRNA level of the pore-forming protein perforin in mitogen-stimulated primary peripheral blood human T cells. In situ hybridization was performed using sense and antisense ribonucleotide probes specific for this granule mediator. After IL-2 treatment, an increase in perforin mRNA could be detected by 4 h; they peaked at 12 h, and decreased after 24 h. The perforin mRNA was also induced in T cells treated with a combination of phorbol ester PMA plus lectin or OKT3 mAb. This latter induction followed slower kinetics, peaking at 48 h. For all three mitogens used, even at peak induction times less than 10% of T cells were labeled with perforin probe. Similar patterns of mRNA expression were observed for both unprimed T cells and lectin-primed T blasts. The induction response of mRNA due to IL-2 stimulation is probably mediated by the IL-2 receptor p75 chain since its mRNA was upregulated by IL-2 with a kinetics comparable to that associated with an increase of perforin mRNA. The p55 IL-2 receptor chain increased much more slowly than p75.     

Optimization of non-isotopic in situ hybridization on formalin-fixed, paraffin-embedded material using digoxigenin-labelled probes and transgenic tissues.

The sensitivity of non-isotopic in situ hybridization (NISH), particularly on formalin-fixed, paraffin-embedded (FFPE) clinical tissues, has been the subject of controversy. Generally, NISH has been regarded as being less sensitive than radiolabelled procedures, although some reports have contradicted this. Accordingly, tissues from mice which were transgenic for variable amounts of the human alpha-1-antitrypsin gene were used to optimize the NISH procedure and to estimate the sensitivity. This approach showed that prolonged incubation of slides in final substrate resulted in high sensitivity--about 13 kb of target DNA. However, this prolonged incubation crucially depended on achieving minimal non-specific background staining. Many factors affected the degree of background staining, but five were particularly important. First, the method of mounting cut sections onto slides. Second, the length of the probe (ideally less than 400 bp). Third, the procedure for proteolytic digestion. Fourth, the denaturation technique, and fifth, the quality of the dextran sulphate used in the hybridization mix. The optimized protocol showed variable patterns of mRNA distribution in the transgenic mouse livers, while DNA distribution appeared uniform.