Green fluorescent protein expression in germ-line transmitted transgenic zebrafish under a stratified epithelial promoter from keratin8.

A zebrafish cDNA encoding a novel keratin protein was characterized and named keratin8, or krt8. krt8 expression was initiated at 4.5 hr postfertilization, immediately after the time of zygotic genome activation. The expression is limited to a single layer of envelope cells on the surface of embryos and, in later stages, it also appears in the innermost epithelial layer of the anterior- and posteriormost portions of the digestive tract. In adult, its expression was limited to the surface layer of stratified epithelial tissues, including skin epidermis and epithelia of mouth, pharynx, esophagus, and rectum but not in the gastral and intestinal epithelia. By using a 2.2-kb promoter from krt8, several stable green fluorescent protein (gfp) transgenic zebrafish lines were established. All of these transgenic lines displayed GFP expression in tissues mentioned above except for the rectum; therefore, the pattern of transgenic GFP expression is essentially identical to that of the endogenous krt8 mRNAs. krt8-GFP fusion protein was also expressed in zebrafish embryos under a ubiquitous promoter, and the fusion protein was capable of assembling into intermediate filaments only in the epithelia that normally expressed krt8 mRNAs, indicating the specificity of keratin assembly in vivo.     

Direct visualization of glucocorticoid receptor positive cells in the hippocampal regions using green fluorescent protein transgenic mice

The hippocampal formation is a plastic brain structure important for certain types of learning and memory, and also vulnerable to the effects of stress and trauma. Since hippocampal neurons express high levels of corticosteroid receptor, the morphological changes, including alterations in the size of soma, and the length and number of neurites and spines, in response to glucocorticoids released as a result of stress are intriguing. In order to highlight the morphology of neurons that express glucocorticoid receptor (GR), we have generated a transgenic mouse line expressing green fluorescent protein (GFP) under the control of the GR promoter. We found strong green fluorescence in the pyramidal cell layer of the CA1 and CA2 regions and the granule cell layer of the dentate gyrus of the hippocampus in brain sections of the transgenic mice. GFP fluorescence was observed not only in somas, but also in neurites including both dendrites and axons. In dissociated culture, we also observed GFP fluorescence in the soma, neurites including both dendrites and axons, and dendritic spines. Microtubule-associated protein 2 immunopositive pyramidal-shaped neurons clearly showed two different populations, GFP positive and GFP negative neurons. These results indicate that this transgenic mouse line should be useful for live imaging of neuronal structure in animals as well as GR-positive cultured cells using GFP as a specific indicator.     

携带增强型绿色荧光蛋白基因的shRNA真核表达载体的构建

目的：构建携带EGFP的shRNA真核表达载体。 方法: 应用PCR的方法从pBSK/U6质粒上扩增出U6启动子及其下游的Xbal、SalI和BamHI酶切位点，并增加NotI位点。将扩增的产物连入pEGFP-C1载体MluI位点处，构建成携带EGFP的siRNA真核表达载体。应用该载体介导针对EGFP的短发夹环RNA（pEGFP/U6/EGFP），转染U251细胞，通过荧光显微镜和流式细胞仪检测EGFP的表达，验证所构建载体在细胞内产生RNA干扰的效果。 结果: 与对照组比较pEGFP/U6/EGFP在细胞内对EGFP的抑制效果达89.8%。 结论: 成功构建携带EGFP的shRNA真核表达载体。     

Zebrafish tiggy-winkle hedgehog promoter directs notochord and floor plate green fluorescence protein expression in transgenic zebrafish embryos.

Zebrafish tiggy-winkle hedgehog (twhh) is a member of the hedgehog gene family that plays an important role in patterning brain, neural tube, somites, and eyes. To better understand the regulation of its tissue-specific expression, the activity of the twhh promoter was determined in zebrafish embryos by transient and transgenic expression analysis. Transient expression studies revealed that the 5.2-kb twhh promoter drove green fluorescence protein (GFP) expression in the notochord, floor plate, and branchial arches. Deletion analysis showed that distinct regions of the twhh promoter regulated the respective notochord or floor plate specific expression. To confirm the tissue specificity of the twhh promoter, transgenic zebrafish containing the twhh-GFP transgene were generated. GFP expression was analyzed in the F1, F2, and F3 generations of the transgenic embryos. The results confirmed the tissue-specific expression of the transgene in the notochord, floor plate, and branchial arches. In addition, GFP expression was also found in the pectoral fin buds, retina, and epithelial lining cells of the Kupffer's vesicle in the transgenic fish embryos. The expression pattern of the twhh-GFP transgene mimicked the expression of the endogenous twhh mRNAs in the floor plate, fin buds, branchial arches, retina, and epithelial lining cells of the Kupffer's vesicle. The expression in the notochord, however, did not mimic the pattern of the endogenous twhh expression. To determine whether no tail (ntl) or floating head (flh) mutants that have developmental defect in the notochord or the Kupffer's vesicle may affect the GFP expression in these regions, GFP expression was analyzed in ntl or flh transgenic embryos. No GFP expression could be detected in the midline region of the ntl transgenic embryos. However, in flh transgenic embryos, although GFP expression was affected in the midline region, its expression in the Kupffer's vesicle appeared normal. Together, these data indicated that the 5.2-kb twhh promoter contains regulatory elements for tissue-specific expression of twhh in the floor plate, pectoral fin bud, branchial arches, retina, and Kupffer's vesicle.     

Destabilized green fluorescent protein for monitoring transient changes in mycobacterial gene expression

The green fluorescent protein (GFP) is a useful reporter for the study of gene expression and protein localisation within living cells. The stability of GFP permits its intracellular accumulation and detection, but renders it less useful for assessing transient changes in gene expression. We have developed a destabilized form of GFP for monitoring gene expression in mycobacteria. By fusing to the C-terminal end of GFP an 11 amino acid peptide encoded by the E. coli ssrA gene, we have developed a form of GFP that exhibits gradual, time-dependent degradation within the fast-growing species Mycobacterium smegmatis. This unstable variant of GFP detected transient changes in the activity of the stress-induced Mycobacterium tuberculosis sigE promoter; by contrast, unmodified GFP only detected a delayed 'switch-on' of this promoter upon exposure to acid stress. Both forms of the protein displayed equivalent stability in the slow-growing species Mycobacterium bovis bacille Calmette-Guerin (BCG), suggesting differing recognition of the ssrA-encoded peptides in slow- and fast-growing mycobacteria. This system will facilitate studies exploring dynamic changes in mycobacterial gene expression.     

Transgenic zebrafish expressing green fluorescent protein in dopaminergic neurons of the ventral diencephalon

We have generated a line of transgenic zebrafish, Tg(dat:EGFP), in which the green fluorescent protein (GFP) is expressed under the control of cis-regulatory elements of the dopamine transporter (dat) gene. In Tg(dat:EGFP) fish, dopamine (DA) neurons are labeled with GFP, including those in ventral diencephalon (vDC) clusters, amacrine cells in the retina, in the olfactory bulb, in the pretectum, and in the caudal hypothalamus. In the vDC, DA neurons of groups 2-6 are correctly labeled with GFP, based on colocalization analyses. MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) treatments induced a modest but significant loss of DA neurons in groups 2-6 of the vDC. This transgenic line will be useful for the study of DA neuron development and in models of DA neuron loss.     

435-bp liver regulatory sequence in the liver fatty acid binding protein (L-FABP) gene is sufficient to modulate liver regional expression in transgenic zebrafish.

Liver fatty acid binding protein (L-FABP) is a small protein that is thought to play an important role in the intracellular binding and trafficking of long chain fatty acids in the liver. Expression of the gene encoding the zebrafish liver fatty acid binding protein is regulated by a 435-bp distal region (-1944 to -1510) of the L-FABP promoter. The 435-bp sequence is sufficient for gene activation in the liver primordia (or bud) and continues to be active in the adult liver when positioned adjacent to the SV40 basal promoter and linked directly to green fluorescent protein. The 435-bp sequence region has two distinct liver regulatory elements, A (-1944 to -1623) and B (-1622 to -1510), and contains multiple putative consensus binding sites. The element A sequence includes two consensus HFH and one HNF-1alpha site and the element B sequence includes one consensus HNF-3beta site. Deletion of an internal 435-bp fragment (-1944 to -1510) including the A and B elements totally ablated the liver-specific activity of the zebrafish L-FABP gene promoter. Deletion of either of the two elements reduces the liver activity. Mutation of the HNF-1alpha site or either of the two HFH sites in the A element or the HNF-3beta site in the B element significantly altered specificity in the liver primordia of transient expression embryos. The importance of the HNF-1alpha consensus binding site in the A element and the HNF-3beta consensus binding site in the B element within the 435-bp distal region of the L-FABP promoter region suggests that combinatorial interactions between multiple regulatory factors are responsible for the gene expression of L-FABP in the liver.     

绿色荧光蛋白与H-2K~b融合基因的构建及表达

目的　构建GFP与H 2Kb 融合基因 ,并分析融合基因在活细胞中的表达。方法　应用基因工程技术构建H 2Kb 与绿色荧光蛋白 (GFP)的融合基因。RT PCR和Western印迹分析基因表达 ;激光共聚焦荧光显微镜观察活细胞内荧光布局。结果　RT PCR和Western印迹分析表明融合基因获得了正确表达。激光共聚焦荧光显微镜观察结果表明 ,Kb GFP融合分子象天然Kb 分子一样表达在细胞膜表面 ,且胞外区域可被Kb 分子特异性单克隆抗体所识别 ,表明Kb GFP融合分子可在细胞内正确折叠和装配。Kb GFP融合基因的瞬间和稳定表达均获得了相同结果。结论　Kb GFP融合蛋白具有GFP的自发荧光特性 ,且不影响Kb 分子在细胞内的正确表达。     

Cryptococcus neoformans differential gene expression detected in vitro and in vivo with green fluorescent protein

Synthetic green fluorescent protein (GFP) was used as a reporter to detect differential gene expression in the pathogenic fungus Cryptococcus neoformans. Promoters from the C. neoformans actin, GAL7, or mating-type alpha pheromone (MFalpha1) genes were fused to GFP, and the resulting reporter genes were used to assess gene expression in serotype A C. neoformans. Yeast cells containing an integrated pACT::GFP construct demonstrated that the actin promoter was expressed during vegetative growth on yeast extract-peptone-dextrose medium. In contrast, yeast cells containing the inducible GAL7::GFP or MFalpha1::GFP reporter genes expressed significant GFP activity only during growth on galactose medium or V-8 agar, respectively. These findings demonstrated that the GAL7 and MFalpha1 promoters from a serotype D C. neoformans strain function when introduced into a serotype A strain. Because the MFalpha1 promoter is induced by nutrient deprivation and the MATalpha locus containing the MFalpha1 gene has been linked with virulence, yeast cells containing the pMFalpha1::GFP reporter gene were analyzed for GFP expression in the central nervous system (CNS) of immunosuppressed rabbits. In fact, significant GFP expression from the MFalpha1::GFP reporter gene was detected after the first week of a CNS infection. These findings suggest that there are temporal, host-specific cues that regulate gene expression during infection and that the MFalpha1 gene is induced during the proliferative stage of a CNS infection. In conclusion, GFP can be used as an effective and sensitive reporter to monitor specific C. neoformans gene expression in vitro, and GFP reporter constructs can be used as an approach to identify a novel gene(s) or to characterize known genes whose expression is regulated during infection.     

组织因子途径抑制因子/绿色荧光蛋白双顺反子真核表达载体构建及其在NIH 3T3细胞中表达

目的构建含人组织因子途径抑制因子(TFPI)和绿色荧光蛋白(GFP)基因的双顺反子真核表达载体,并验证其在NIH 3T3细胞中的表达,为血管再狭窄的防治提供一个具有示踪和治疗双重作用的有效载体。方法以含有全长cDNA的pIRES-TFPI为模板,多聚酶链反应(PCR)扩增TFPI全长cDNA,经酶切后插入到pIRES2-AcGFP1-Nuc载体中,构建成双顺反子真核表达载体,重组质粒经酶切图谱分析、PCR扩增及测序鉴定后命名为pIRES2-AcGFP1-Nuc-TFPI。将其转染NIH3T3细胞,采用荧光显微镜观察GFP在细胞中的表达,以RT-PCR检测TFPI在细胞内的表达。结果经酶切图谱分析、PCR扩增及DNA测序证实双顺反子真核表达载体构建正确;荧光显微镜可观察到细胞内GFP的表达;RT-PCR证实经TFPI基因转染的细胞内TFPI mRNA表达增高。结论成功构建了包括TFPI和GFP的真核双表达载体,并使其在NIH3T3细胞中顺利表达。     

Preparation and observation of fresh-frozen sections of the green fluorescent protein transgenic mouse head

Hard tissue decalcification can cause variation in the constituent protein characteristics. This paper describes a method of preparating of frozen mouse head sections so as to clearly observe the nature of the constituent proteins. Frozen sections of various green fluorescent protein (GFP) transgenic mouse heads were prepared using the film method developed by Kawamoto and Shimizu. This method made specimen dissection without decalcification possible, wherein GFP was clearly observed in an undamaged state. Conversely, using the same method with decalcification made GFP observation in the transgenic mouse head difficult. This new method is suitable for observing GFP marked cells, enabling us to follow the transplanted GFP marked cells within frozen head sections.     

携EGFP的人Synoviolin基因慢病毒表达载体的构建与鉴定

目的构建携EGFP的人Synoviolin基因慢病毒表达载体。方法应用基因重组手段,SalⅠ/NotⅠ双酶切质粒pIRES2-EGFP-syno,得到含EGFP和人Synoviolin的基因片段,将其亚克隆至入门载体pENTR1A的多克隆位点内得到入门质粒pENTR1A-syno-egfp。采用LR重组酶将pENTR1A-syno-egfp和目的载体pLenti4/TO/V5-DEST进行重组反应,形成慢病毒表达载体pLenti4/TO/V5-DEST-syno-egfp。将pLenti4/TO/V5-DEST-syno-egfp与包装质粒混合,利用脂质体共转染293FT细胞,包装产生慢病毒,以293FT细胞GFP蛋白的表达水平测定病毒滴度。结果PCR,酶切及测序结果表明慢病毒表达载体pLenti4/TO/V5-DEST-syno-egfp构建成功,转染后的293FT细胞在荧光显微镜下观察可见强绿色荧光。包装的慢病毒原液滴度为1×10~8TU/ml。结论成功构建了EGFP和Synoviolin基因共表达的慢病毒表达载体,为Synoviolin基因的功能研究提供了高效稳定的转基因技术平台。     

用小双链RNA抑制转化入HUVEC中绿色荧光蛋白基因的表达

目的：建立稳定表达绿色荧光蛋白(GFP)的人脐静脉血管内皮细胞（HUVECs），研究小干扰RNA(siRNA)对HUVECs中GFP表达的抑制作用。 方法： 用lipofectamine2000将编码GFP的质粒pN3-EGFP转入HUVECs中。用G418筛选并维持已转化pN3-EGFP的HUVEC(HUVEC-GFP)。利用T7 RNA转录试剂盒，制备可抑制GFP基因表达的siRNA（GFPsiRNA）和无关对照的RNA（control siRNA）。用oligofectamine将siRNA转入HUVEC-GFP中。继续培养48 h后，检测HUVEC-GFP中GFP蛋白和mRNA表达水平。 结果： 用G418筛选获得了HUVEC-GFP细胞株，可以观察到GFP的稳定表达。HUVEC-GFP转化siRNA后48 h，GFP的荧光强度明显下降，而对照组的荧光强度无明显下降。半定量RT-PCR检测显示，GFPsiRNA对GFP mRNA表达有较强的抑制作用，抑制率达40%，而control siRNA对GFP mRNA表达水平无明显的抑制作用。 结论： 利用体外转录合成的siRNA能有效地抑制HUVECs中GFP的表达。     

Expression analysis of green fluorescent protein in retinal neurons of four transgenic mouse lines

Transgenic mice that express enhanced green fluorescent protein (EGFP) under the control of a cell-specific promoter have been used with great success to identify and label specific cell types of the retina. We studied the expression of EGFP in the retina of mice making use of four transgenic mouse lines. Expression of EGFP driven by the calretinin promoter was found in amacrine, displaced amacrine and ganglion cells. Comparison of the EGFP expression and calretinin immunolabeling showed that many but not all cells appear to be double labeled. Expression of EGFP under the control of the choline acetyltransferase promoter was found in amacrine cells; however, the cells did not correspond to the well known cholinergic (starburst) cells of the mouse retina. The expression of EGFP under the control of the parvalbumin promoter was restricted to amacrine cells of the inner nuclear layer and to cells of the ganglion cell layer (displaced amacrine cells and ganglion cells). Most of the cells were also immunoreactive for parvalbumin, however, differences in labeling intensity were observed. The expression of EGFP driven by the promoter for the 5-HT3 A receptor (5-HTR3A) was restricted to type 5 bipolar cells. In contrast, immunostaining for 5-HTR3A was found in synaptic hot spots in sublamina 1 of the inner plexiform layer and was not related to type 5 bipolar cells. The results show that these transgenic mice are very useful for future electrophysiological studies of specific types of amacrine and bipolar cells that express EGFP and thus permit directed microelectrode targeting under microscopic control.