Characterization of transgenic zebrafish lines that express GFP in the retina,pineal gland,olfactory bulb,hatching gland,and optic tectum

Transgenic animals are powerful tools to study gene function invivo. Here we characterize several transgenic zebrafish lines that express green fluorescent protein (GFP) under the control of the LCR^RH2-RH2-1 or LCR^RH2-RH2-2 green opsin regulatory elements. Using confocal immunomicroscopy, stereo-fluorescence microscopy, and Western blotting, we show that the Tg(LCR^RH2-RH2-1:GFP)^pt112 and Tg(LCR^RH2-RH2-2:GFP)^pt115 transgenic zebrafish lines express GFP in the pineal gland and certain types of photoreceptors. In addition, some of these lines also express GFP in the hatching gland, optic tectum, or olfactory bulb. Some of the expression patterns differ significantly from previously published similar transgenic fish lines, making them useful tools for studying the development of the corresponding tissues and organs. In addition, the variations of GFP expression among different lines corroborate the notion that transgenic expression is often subjected to position effect, thus emphasizing the need for careful verification of expression patterns when transgenic animal models are utilized for research.     

受强力霉素调节的自杀基因表达载体系统的构建和在乳腺癌细胞株(MCF-7)的表达

自杀基因治疗是恶性肿瘤基因治疗中最常用的途径之一,以递转录病毒载体-pRevTRE为基础进行载体构建,首先使用PCR技术对单纯疱疹病毒胸苷激酶基因（HSVtk)进行扩增,将HSVtk基因插入到pRe-vTRE,形成重组载体pRevTRE/HSVtk,用磷酸钙共沉淀法,经过两轮转染,分别将pRevTRE/HSVtk和pRevTet-On质粒导入乳腺癌细胞株（MCF-7）经过潮霉素B（HygromycinB)和G418筛选,建立了一株稳定的受四环素衍生物-强力霉素（Doxycycline,Dox)调控,表达HSVtk基因产物的人乳腺癌细胞株MCF/TRE/tk/Tet-On,HSVtk基因表达产物可以将无毒性的药物前体Ganciclovir(GCV)转变成一种有毒的代谢产物,从而杀死乳腺癌细胞株（MCF-7）,达到基因治疗的目的。     

Generation and Characterization of a Transgenic Zebrafish Expressing the Reverse Tetracycline Transactivator

Conditional expression of a target gene during zebrafish development is a powerful approach to elucidate gene functions. The tetracycline-controlled systems have been successfully used in the modulation of gene expression in mammalian cells, but few lines of zebrafish carrying these systems are currently available. In this study, we had generated a stable transgenic zebrafish line that ubiquitously expressed the second-generation of reverse Tet transactivator （rtTA-M2）. Southern blotting analysis and high-throughput genome sequencing verifed that a single copy of rtTA-M2 gene had stably integrated into the zebrafish genome. After induction with doxycycline （Dox）, a strong green fluorescent protein （GFP） was seen in rtTA-transgenic eggs injected with pTRE--EGFP plasmids. The fluorescent signal gradually decreased after the withdrawal of Dox and disappeared. However, leaky expression of GFP was undetectable before Dox- induction. Additionally, transgenic embryos expressing rtTA-M2 exhibited no obvious defects in morphological phenotypes, hatching behavior and expression patterns of developmental marker genes, suggesting that rtTA-M2 had little effect on the development of transgenic zebrafish. Moreover, expressed Dickkopf-1 （DKK1） in pTRE-DKKl-injected embryos led to alterations in the expression of marker genes associated with Wnt signaling. Thus, this rtTA-transgenic zebrafish can be utilized to dissect functions of genes in a temporal manner.     

Development of a transgenic zebrafish model expressing GFP in the notochord,somite and liver directed by the <Emphasis Type="Italic">hfe2</Emphasis> gene promoter

Hemojuvelin, also known as RGMc, is encoded by hfe2 gene that plays an important role in iron homeostasis. hfe2 is specifically expressed in the notochord, developing somite and skeletal muscles during development. The molecular regulation of hfe2 expression is, however, not clear. We reported here the characterization of hfe2 gene expression and the regulation of its tissue-specific expression in zebrafish embryos. We demonstrated that the 6 kb 5′-flanking sequence upstream of the ATG start codon in the zebrafish hfe2 gene could direct GFP specific expression in the notochord, somites, and skeletal muscle of zebrafish embryos, recapitulating the expression pattern of the endogenous gene. However, the Tg(hfe2:gfp) transgene is also expressed in the liver of fish embryos, which did not mimic the expression of the endogenous hfe2 at the early stage. Nevertheless, the Tg(hfe2:gfp) transgenic zebrafish provides a useful model to study liver development. Treating Tg(hfe2:gfp) transgenic zebrafish embryos with valproic acid, a liver development inhibitor, significantly inhibited GFP expression in zebrafish. Together, these data indicate that the tissue specific expression of hfe2 in the notochord, somites and muscles is regulated by regulatory elements within the 6 kb 5′-flanking sequence of the hfe2 gene. Moreover, the Tg(hfe2:gfp) transgenic zebrafish line provides a useful model system for analyzing liver development in zebrafish.     

Delayed and Restricted Expression of UAS-Regulated GFP Gene in Early Transgenic Zebrafish Embryos by Using the GAL4/UAS System

A stable Tg(UAS:GFP) zebrafish line was generated and crossed with Tg(hsp70:GAL4) line, in which the GAL4 gene is under the control of an inducible zebrafish promoter derived from the heat shock 70 protein gene (hsp70). The dynamic green fluorescent protein (GFP) expression in early zebrafish embryos in the GAL4/UAS binary system was then investigated. We found that, at early developmental stages, expression of GFP effector gene was restricted and required a long recovery time to reach a detectable level. At later developmental stage (after 2 days postfertilization), GFP could be activated in multiple tissues in a shorter time, apparently due to a higher level of GAL4 messenger RNA induction. It appears that the type of tissues expressing GFP was dependent on whether they had been developed at the time of heat shock. Therefore, the delayed and restricted transgene expression should be taken into consideration when GAL4/UAS system is used to study transgene expression in early developmental stages.     

A novel adenoviral vector carrying an all-in-one Tet-On system with an autoregulatory loop for tight,inducible transgene expression

Background

One of the most commonly used vectors for gene therapy is the adenoviral vector; its ability to tightly regulate transgene expression is critical for optimizing therapeutic outcomes. The tetracycline-regulated system (especially the Tet-On system) for gene expression is one of the most valuable tools for controlling gene expression. The major problem of an adenoviral vector carrying a Tet-On system is suboptimal regulation of transgene expression.

Results

We constructed a single adenoviral vector carrying in its E1 region a novel “all-in-one” Tet-On system with an autoregulatory loop. This system had improved Dox-inducible gene expression in terms of low basal expression, high induced expression and high responsiveness to Dox. To our knowledge, this is the first reported adenovirus-based, all-in-one Tet-On system with an autoregulatory loop inserted into a single region of adenoviral genome. This system was further tested by inducible expression of soluble tumor necrosis factor-related apoptosis-inducing ligand (sTRAIL). The adenovirus that expressed soluble TRAIL under the control of this novel Tet-On system showed tumor-derived cells inhibitory activity in SW480 cells only under induced conditions.

Conclusions

Our novel, single adenoviral vector carrying in its E1 region an all-in-one Tet-On system with an autoregulatory loop displayed tight regulation of transgene expression in vitro. This system has great potential for a variety of applications, including gene therapy and the study of gene function.     

Inducible and Reversible Lentiviral and Recombination Mediated Cassette Exchange (RMCE) Systems for Controlling Gene Expression

Manipulation of gene expression to invoke loss of function (LoF) or gain of function (GoF) phenotypes is important for interrogating complex biological questions both in vitro and in vivo. Doxycycline (Dox)-inducible gene expression systems are commonly used although success is often limited by high background and insufficient sensitivity to Dox. Here we develop broadly applicable platforms for reliable, tightly controlled and reversible Dox-inducible systems for lentiviral mediated generation of cell lines or FLP Recombination-Mediated Cassette Exchange (RMCE) into the Collagen 1a1 (Col1a1) locus (FLP-In Col1a1) in mouse embryonic stem cells. We significantly improve the flexibility, usefulness and robustness of the Dox-inducible system by using Tetracycline (Tet) activator (Tet-On) variants which are more sensitive to Dox, have no background activity and are expressed from single Gateway-compatible constructs. We demonstrate the usefulness of these platforms in ectopic gene expression or gene knockdown in multiple cell lines, primary neurons and in FLP-In Col1a1 mouse embryonic stem cells. We also improve the flexibility of RMCE Dox-inducible systems by generating constructs that allow for tissue or cell type-specific Dox-inducible expression and generate a shRNA selection algorithm that can effectively predict potent shRNA sequences able to knockdown gene expression from single integrant constructs. These platforms provide flexible, reliable and broadly applicable inducible expression systems for studying gene function.     

Tet-On调控HSVtk表达的重组腺相关病毒载体的构建和感染活性的检测

构建含有Tet基因调节系统及自杀基因HSVtk的重组腺相关病毒载体pAAV TRE HSVtk Tet_On ,并使用PCR技术和限制性内切酶消化进行鉴定。用构建好的重组质粒分别与辅助质粒pAAV_RC、pHelper以磷酸钙共沉淀法转染HEK2 93细胞,进行病毒包装后得到了AAV TRE HSVtk Tet_On重组腺相关病毒,以氯化铯密度梯度离心对包装好的病毒进行纯化。用纯化后的重组腺相关病毒感染乳腺癌细胞株MCF_7后,斑点杂交检测结果显示,HSVtk基因整合进入MCF_7细胞基因组中。有感染活性的重组腺相关病毒能将目的基因转移到宿主细胞中,在Dox诱导下,GCV对AAV感染的MCF_7细胞具有明显的杀伤作用。     

Maize Ac/Ds transposon system leads to highly efficient germline transmission of transgenes in medaka (Oryzias latipes)

As the medaka is a popular fish model in genetics, developmental biology and toxicology, the development of an efficient transgenic medaka technique is important for a variety of biological experiments. Here we demonstrated that the maize transposon system, Ac/Ds, greatly improved the transgenesis of microinjected DNA. Using the Ac/Ds system, two types of stable transgenic medaka lines, Tg(hsp70:gfp) and Tg(cyp1a1:gfp), were established with germline transmission rates of 83.3% (10/12) and 100.0% (4/4) from GFP-expressing founders, respectively. The percentages of transgenic progeny ranged between 3.1% and 100.0% in F1 from different transgenic founders. Interestingly, multiple insertions were found from transgenic founders and the cloned insertion sites confirmed the transposition mediated by Ac transposase. In addition, we demonstrated the inducible GFP expression in both GFP transgenic medaka lines. In Tg(hsp70:gfp) whose gfp gene was under the control of a heat shock inducible medaka hsp70 promoter, GFP expression was induced ubiquitously after heat shock. In Tg(cyp1a1:gfp), the gfp gene was driven by medaka cyp1a1 promoter that could be activated by various xenobiotic chemicals including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD); indeed, GFP expression was found to be induced in the liver, intestine and kidney by TCDD. Our data presented here demonstrated the highly efficient transgenesis with the aid of the maize Ac/Ds transposon system.     

Evolution and Functional Characterisation of Melanopsins in a Deep-Sea Chimaera (Elephant Shark,Callorhinchus milii)

Non-visual photoreception in mammals is primarily mediated by two splice variants that derive from a single melanopsin (OPN4M) gene, whose expression is restricted to a subset of retinal ganglion cells. Physiologically, this sensory system regulates the photoentrainment of many biological rhythms, such as sleep via the melatonin endocrine system and pupil constriction. By contrast, melanopsin exists as two distinct lineages in non-mammals, opn4m and opn4x, and is broadly expressed in a wide range of tissue types, including the eye, brain, pineal gland and skin. Despite these findings, the evolution and function of melanopsin in early vertebrates are largely unknown. We, therefore, investigated the complement of opn4 classes present in the genome of a model deep-sea cartilaginous species, the elephant shark (Callorhinchus milii), as a representative vertebrate that resides at the base of the gnathostome (jawed vertebrate) lineage. We reveal that three melanopsin genes, opn4m1, opn4m2 and opn4x, are expressed in multiple tissues of the elephant shark. The two opn4m genes are likely to have arisen as a result of a lineage-specific duplication, whereas “long” and “short” splice variants are generated from a single opn4x gene. By using a heterologous expression system, we suggest that these genes encode functional photopigments that exhibit both “invertebrate-like” bistable and classical “vertebrate-like” monostable biochemical characteristics. We discuss the evolution and function of these melanopsin pigments within the context of the diverse photic and ecological environments inhabited by this chimaerid holocephalan, as well as the origin of non-visual sensory systems in early vertebrates.     

Generation of living color transgenic zebrafish to trace somatostatin-expressing cells and endocrine pancreas organization

In the present study, both gfp and rfp transgenic zebrafish lines using a 2.5-kb zebrafish somatostain2 (sst2) promoter were generated. During embryonic development, expression of GFP/RFP in the endocrine pancreas of transgenic embryos was initiated at ∼20 hpf and the number of GFP/RFP positive cells in the pancreas increased in subsequent stages; thus, our newly generated Tg(sst2:gfp) and Tg(sst2:rfp) lines faithfully recapitulated sst2 expression in endocrine pancreatic cells and provided a useful tool in analyzing the development of Sst2-producing δ-cells in the pancreas. By crossing these new transgenic lines with previously available transgenic lines targeted in insulin (Ins)-producing β-cells, Tg(ins:gfp) and Tg(ins:rfp), in combination with immunodetection of glucagon (Gcg)-producing α-cells and pancreatic polypeptide (PP)-producing PP-cells, the organization and composition of endocrine islets were investigated in both embryonic and adult pancreas. We found that there was always a big cluster of endocrine cells (principal islet) in the anterior-dorsal pancreas, followed by numerous smaller clusters (variable in size) of endocrine cells (secondary islets) along the anterior–posterior axis of the pancreas. All four types of endocrine cells were found in the principal islet, but secondary islets may or may not contain PP-cells. In addition, there were also discrete endocrine cells throughout the pancreas. In all co-localization experiments, we did not find any endocrine cells positive for more than one hormone markers, suggesting that these endocrine cells produce only a single hormone. In both principal and secondary islets, we found that β-cells were generally located in the center and non-β cells in the periphery; reminiscent of the “mantel–core” organization of islets of Langerhans in mammals where β-cells form the core and non-β-cells the mantel. In zebrafish primary islet, β-cells constitute most of the mass (∼50%), followed by δ-cells and α-cells (20–25% each), and PP-cells (1–2%); this is also similar to the composition of mammalian islets.     

The effect of trehalose on autophagy-related proteins and cyst growth in a hypomorphic Pkd1 mouse model of autosomal dominant polycystic kidney disease

Autosomal dominant polycystic kidney disease (ADPKD) is a common inherited disorder characterized by kidney cyst growth often resulting in end-stage renal disease. There is growing attention on understanding the role of impaired autophagy in ADPKD. Trehalose (TRE) has been shown to increase both protein stability and aggregate clearance and induce autophagy in neurodegenerative diseases. TRE treatment in wild type mice compared to vehicle resulted in increased expression in the kidney of Atg12–5 complex and increased Rab9a, autophagy-related proteins that play a role in the formation of autophagosomes. Thus, the aim of the study was to determine the effect of TRE on cyst growth and autophagy-related proteins, in the hypomorphic Pkd1^RC/RC mouse model of ADPKD. Pkd1^RC/RC mice were treated 2% TRE in water from days 50 to 120 of age. TRE did not slow cyst growth or improve kidney function or affect proliferation and apoptosis in Pkd1^RC/RC kidneys. In Pkd1^RC/RC vs. wild type kidneys, expression of the Atg12–5 complex was inhibited by TRE resulting in increased free Atg12 and TRE was unable to rescue the deficiency of the Atg12–5 complex. Rab9a was decreased in Pkd1^RC/RC vs. wild type kidneys and unaffected by TRE. The TRE-induced increase in p62, a marker of autophagic cargo, that was seen in normal kidneys was blocked in Pkd1^RC/RC kidneys. In summary, the autophagy phenotype in Pkd1^RC/RC kidneys was characterized by decreases in crucial autophagy-related proteins (Atg12–5 complex, Atg5, Atg16L1), decreased Rab9a and increased mTORC1 (pS6^S240/244, pmTOR^S2448) proteins. TRE increased Atg12–5 complex, Rab9a and p62 in normal kidneys, but was unable to rescue the deficiency in autophagy proteins or suppress mTORC1 in Pkd1^RC/RC kidneys and did not protect against cyst growth.     

Oxidative Stress and Regulation of Pink1 in Zebrafish (Danio rerio)

Oxidative stress-mediated neuronal dysfunction is characteristic of several neurodegenerative disorders, including Parkinson’s disease (PD). The enzyme tyrosine hydroxylase (TH) catalyzes the formation of L-DOPA, the rate-limiting step in the biosynthesis of dopamine. A lack of dopamine in the striatum is the most characteristic feature of PD, and the cause of the most dominant symptoms. Loss of function mutations in the PTEN-induced putative kinase (PINK1) gene cause autosomal recessive PD. This study explored the basic mechanisms underlying the involvement of pink1 in oxidative stress-mediated PD pathology using zebrafish as a tool. We generated a transgenic line, Tg(pink1:EGFP), and used it to study the effect of oxidative stress (exposure to H₂O₂) on pink1 expression. GFP expression was enhanced throughout the brain of zebrafish larvae subjected to oxidative stress. In addition to a widespread increase in pink1 mRNA expression, mild oxidative stress induced a clear decline in tyrosine hydroxylase 2 (th2), but not tyrosine hydroxylase 1 (th1) expression, in the brain of wild-type larvae. The drug L-Glutathione Reduced (LGR) has been associated with anti-oxidative and possible neuroprotective properties. Administration of LGR normalized the increased fluorescence intensity indicating pink1 transgene expression and endogenous pink1 mRNA expression in larvae subjected to oxidative stress by H₂O₂. In the pink1 morpholino oliogonucleotide-injected larvae, the reduction in the expression of th1 and th2 was partially rescued by LGR. The pink1 gene is a sensitive marker of oxidative stress in zebrafish, and LGR effectively normalizes the consequences of mild oxidative stress, suggesting that the neuroprotective effects of pink1 and LGR may be significant and useful in drug development.