应用2A肽策略构建双基因共表达转基因斑马鱼的研究

目的以Tol2为骨架载体,以绿色荧光蛋白(GFP)、Cherry为报告基因,探讨采用2A肽双基因载体构建策略构建单启动子双基因共表达质粒的方法;将B细胞刺激因子(BAFF)分别置于2A序列前后位置,分析位置效应对跨膜融合蛋白的表达与剪切的影响,探讨多基因共表达转基因斑马鱼构建技术。方法以In Fusion法将GFP-2A-Cherry序列构建到Tol2质粒上,所得p Tol-GFP-2A-Cherry质粒转染He La细胞、显微注射1-细胞期斑马鱼受精卵;倒置荧光显微镜观察He La细胞、斑马鱼幼鱼体内GFP与Cherry蛋白的表达,Western blot法验证GFP和Cherry蛋白的表达量与剪切情况;分别构建p Tol2-GFP-2A-BAFF与p Tol2-BAFF-2A-Cherry质粒,Western blot法检查BAFF的表达与剪切情况。结果 p Tol2-GFP-2A-Cherry质粒转染的He La细胞,GFP与Cherry均可单独表达且表达呈现时空一致性;GFP-2A-Cherry融合蛋白可被剪切为GFP与Cherry,且成等比例表达趋势。p Tol2-GFP-2A-Cherry质粒显微注射1-细胞期斑马鱼受精卵可获得可单独表达GFP与Cherry蛋白的转基因斑马鱼;p Tol2-GFP-2A-BAFF与p Tol2-BAFF-2A-Cherry于斑马鱼体内均有融合蛋白的表达,且BAFF序列位于2A序列后更易于融合蛋白的剪切。结论通过2A肽策略构建可实现在斑马鱼体内单一载体、单一启动子调控双基因表达目的。发现编码跨膜分泌蛋白的功能基因位于2A序列的不同位置会直接影响蛋白的剪切,功能基因位于2A序列后易于跨膜蛋白的剪切。     

过表达Baff转基因斑马鱼的构建

目的体外克隆斑马鱼baff基因并分别构建pEGFP-C1-baff、pEGFP-N1-baff、pIRES2-EGFP-baff重组质粒,通过胚胎显微注射获得过表达baff的转基因斑马鱼,以探讨其作为人类SLE模型和用于SLE药物筛选的意义。方法通过RT-PCR法由斑马鱼脾脏克隆出斑马鱼baff基因全长807 bp蛋白编码区域,分别构建baff过表达载体pEGFP-C1-baff,pEGFP-N1-baff及pIRES2-EGFP-baff重组质粒,体外细胞转染并通过免疫印迹法验证蛋白表达后,通过胚胎显微注射过表达载体,GFP荧光跟踪并筛选阳性鱼。结果通过体外细胞转染实验与免疫印迹法验证了pEGFP-C1-baff,pEGFP-N1-baff转染后细胞Baff-GFP融合蛋白的成功表达,通过胚胎显微注射与GFP荧光筛选,成功获得过表达baff的转基因斑马鱼。结论本研究所构建pEGFP-C1-baff、pEGFP-N1-baff、pIRES2-EGFP-baff重组质粒均可通过显微注射获得过表达baff的阳性转基因斑马鱼,为进一步探讨其作为人类SLE疾病模型的意义及Baff拮抗药物的筛选奠定基础。     

Baff转基因斑马鱼的构建及相关基因表达检测

通过RT-PCR法由斑马鱼脾脏克隆B细胞刺激因子baff基因,构建过表达斑马鱼baff且携带有绿色荧光标记蛋白的重组质粒pIRES2-GFP-baff;胚胎显微注射获得转基因斑马鱼胚胎;通过GFP荧光标记跟踪并筛选转基因阳性鱼;Western blot法鉴定Baff-GFP融合蛋白表达情况;qPCR检测baff,GFP及baff下游相关基因bcl-2,il-4mR-NA表达情况。结果表明细胞、胚胎及幼鱼baff和GFP均高表达,baff下游基因bcl-2激活和il-4基因抑制表达。通过胚胎显微注射法可成功获得过表达baff的转基因斑马鱼,此研究为建立红斑狼疮转基因斑马鱼模型及高通量筛选Baff拮抗剂奠定了基础。     

ripply1在斑马鱼早期胚胎背腹发育中的作用

目的探究ripply1基因在斑马鱼早期背腹轴发生过程中的作用。方法利用斑马鱼整封原位杂交技术揭示ripply1基因在斑马鱼早期胚胎发育过程中的表达模式,通过显微注射技术在胚胎1细胞期注射ripply1的mRNA来高表达Ripply1蛋白并在后期观察胚胎背腹标记基因的变化及胚胎形态变化。利用Tol2转基因技术构建ripply1启动子驱动的GFP转基因鱼。结果原位杂交结果显示ripply1基因在斑马鱼原肠早期胚盾期特异表达在胚盾处,即预定的背部。高表达ripply1后,在胚盾期背部标记基因表达范围扩大,腹部标记基因表达减弱,受精后24小时胚胎表现出严重的背部化表型:头部增大,腹部卵黄延伸减弱,尾部躯干及尾部区域减少,有的甚至形成了第二个体轴。得到的转基因鱼揭示ripply1母源表达,并且转录起始位点上游1200个碱基驱动的GFP能模拟内源基因的表达图式。结论 ripply1可能参与斑马鱼胚胎早期背腹轴的发生。     

利用Tol2转座子构建转基因草金鱼初探

青鳉Tol2转座子是脊椎动物中发现的第一例天然具有活性的转座子,目前已经被成功应用于多种模式动物的转基因研究中。采用PCR的方法以质粒pCAgcGH为模板亚克隆出一段含有草鱼生长激素(GH)5个外显子、鲤鱼β-actin启动子及多个酶切位点的序列,并将这段序列与经双酶切处理的质粒pTol2-MCS-EGFP进行重组连接,得到重组质粒pTol2-GH-EGFP。采用显微注射的方法将重组质粒pTol2-GH-EGFP与体外合成的Tol2转座酶mRNA一起注入草金鱼受精卵中。通过观察绿色荧光和PCR检验绿色荧光蛋白和GH基因的表达,筛选出成功转入外源基因的草金鱼。阳性表达检出率为17.3%。利用Tol2转座子构建转基因草金鱼,不仅丰富了Tol2转座子的应用范围,而且为进一步利用Tol2转座元件进行观赏鱼转基因及基因表达研究奠定了基础。     

荧光转基因斑马鱼Tg(ttn.2:EGFP)的构建

为了建立一种用于研究肌肉和心脏发育及其相关疾病的绿色荧光蛋白(enhanced green fluorescent protein,EGFP)转基因斑马鱼品系,本研究使用斑马鱼ttn.2基因编码区上游启动子序列和绿色荧光蛋白基因编码序列构建了重组表达载体,并将该载体和Tol2转座酶的加帽mRNA显微共注射入斑马鱼1-细胞期胚胎,通过荧光检测、遗传杂交筛选和分子鉴定等方法,成功建立了能稳定遗传的Tg(ttn.2:EGFP)转基因斑马鱼品系。荧光表达分析及原位杂交分析结果表明,绿色荧光信号在斑马鱼肌肉和心脏组织中特异表达模式与ttn.2基因的mRNA表达一致。通过反向PCR鉴定转基因表达载体在F1代斑马鱼品系中的随机整合位点,结果表明:No.33转基因品系的EGFP基因整合在斑马鱼的4号和11号染色体上,No.34转基因品系则整合在1号染色体上。该荧光转基因斑马鱼品系Tg(ttn.2:EGFP)的成功构建为肌肉和心脏发育以及相关疾病研究提供了一个新的理想实验模型。此外,绿色荧光强烈表达的斑马鱼品系还可以作为一种新的观赏鱼。     

一种微管-荧光融合蛋白嵌合标记斑马鱼运动神经元系统的建立

运动神经元是一类支配运动行为的重要神经元。传统的荧光蛋白标记的转基因斑马鱼品系(用于活体成像分析运动神经元形态发生)存在胞体密集、突触交错、不好区分单个神经元等不足。为了优化活体成像分析运动神经元,本研究旨在建立一种微管-荧光融合蛋白嵌合标记斑马鱼运动神经元系统。首先通过Gateway克隆技术将运动神经元表达基因mnx1启动子序列与绿色荧光蛋白-α-Tubulin融合蛋白序列构建到含有Tol2转座位点的表达载体中,然后将该质粒和Tol2 mRNA同时注射到4～8细胞期斑马鱼受精卵中,在72 hpf (hours post fertilization)进行共聚焦显微成像分析。结果显示,该系统中绿色荧光融合蛋白在3种类型运动神经元中表达,从而实现单个运动神经元嵌合标记。本研究进一步探索注射剂量与嵌合标记神经元数量以及分布频率的关系,并确定了重组蛋白的合适剂量(15 ng)。此外,本研究在该模型上验证了insm1a和kif15表达下调导致的运动神经元异常发育。这些结果表明我们成功建立了一种微管-荧光融合蛋白嵌合标记斑马鱼运动神经元系统,为探究运动神经元的发育和形态发生提供了一个直观和快速的模...     

斑马鱼两种转基因方法的比较

对斑马鱼(Danio rerio)的两种转基因方法进行比较,分别采用显微注射和电脉冲导入法将携带有GFP报告基因的表达载体质粒导入斑马鱼受精卵,得出电脉冲最佳导入条件为最适电压125 V/cm,电阻50 Ω,最佳导入时期为1-2细胞期(40-50 min),以及最佳外源基因浓度为300 ng/μL.利用两种方法均得到转GFP基因的斑马鱼,而两种方法对比的结果表明,显微注射法耗时费力,但转基因阳性率高;电脉冲法一次可以处理大批量受精卵,但转基因阳性率远低于显微注射法.     

四环素诱导肝脏特异表达绿色荧光蛋白转基因斑马鱼模型建立

目的探索tet-on四环素诱导表达系统在斑马鱼体内应用策略与技术路线,构建四环素诱导肝脏特异表达绿色荧光蛋白的转基因斑马鱼,为条件型功能基因研究及组织特异转基因斑马鱼疾病模型的建立奠定基础。方法构建肝脏特异启动子fabp10启动rt TA蛋白表达的重组质粒pfabp10-rt TA,联合p TRE-Tight-BI-Ac GFP1质粒转染He La细胞后给予doxycycline诱导,Western blot法验证;pfabp10-rt TA联合p TRE-Tight-BI-Ac GFP1质粒注射斑马鱼1-细胞期受精卵后,30μg/m L doxycycline诱导,荧光筛选稳定整合个体。结果共转染pfabp10-rt TA与p TRE-Tight-BI-Ac GFP1的He La细胞经1μg/m L浓度doxycycline诱导培养液诱导,GFP表达量显著高于不加doxycycline培养液对照组;筛选获得的稳定整合斑马鱼幼鱼,在浓度为30μg/m L doxycycline条件下,肝脏明显有绿色荧光表达,对照组幼鱼肝脏位置未有明显绿色荧光。结论 Tet-On四环素诱导表达系统可用于建立四环素调控斑马鱼肝脏特异表达外源基因;利用该技术可建立诱导肝脏表达GFP建立转基因斑马鱼品系,为建立条件型转基因斑马鱼疾病模型、探索肝脏器官发生发育等研究提供良好的模式动物工具。     

转基因动物研究现状

导言 1980年Gordon等人将疱疹病毒胸苷激酶基因和SV40早期基因启动子重组到质粒pBR322中,再由显微注射植入小鼠受精卵原核,获得第一只转基因小鼠。原理上任何克隆基因都能永久并入哺乳动物胚胎基因组。显微注射后,外源DNA整合进细胞,随后繁衍成种系。因此通过选育能永久建立携带一个或多个新基因的动物谱系。“转基因”(Transgenic)一词应运而生。在此之前,Jaenisch(1976)观察到逆转录病毒(又称逆病毒)感染小鼠胚胎,使单个前病毒DNA插入小鼠染色体DNA。而后含异源基因的重组感染性逆病毒系统的发展导致了用     

miR-22心肌特异转基因斑马鱼的构建及心肌肥厚的评估

目的:构建miR-22心肌特异转基因斑马鱼系,在体评估miR-22对于心肌肥厚的作用。方法:构建pTol2-CMLC2-miR-22-IRES-EGFP表达载体。通过显微注射的方法将tol2重组质粒于一细胞期注射入斑马鱼受精卵胚胎中,荧光筛选获得心肌特异表达绿色荧光的斑马鱼胚胎,并稳定表达传代。然后对稳定传代的成年斑马鱼心脏进行心肌肥厚及心功能的检测。结果:成功建立了miR-22心肌特异转基因斑马鱼系,通过定量PCR确定心肌中miR-22表达升高,荧光显微镜观察发现斑马鱼心肌出现绿色荧光。miR-22心脏特异过表达的转基因鱼系的成年鱼与野生对照组相比,出现了心肌肥厚的现象,心肌肥厚分子标志物nppa、myh7明显升高。斑马鱼心脏病理切片结果同样显示出miR-22心肌特异转基因斑马鱼出现了心肌肥厚的现象。结论:成功构建了miR-22心肌特异转基因斑马鱼,为研究心肌中miR-22的生物学功能提供了重要的工具,并证明miR-22心脏特异过表达会引起斑马鱼心肌肥厚。     

Transgenic overexpression of BAFF regulates the expression of immune-related genes in zebrafish, <Emphasis Type="Italic">Danio rerio</Emphasis>

The B-cell activating factor (BAFF) is a member of tumour necrosis factor (TNF) superfamily that specifically regulates B lymphocyte proliferation and survival. Excess BAFF leads to overproduction of antibodies for secretion, anti-dsDNA antibodies and a lupus-like syndrome in mice. To investigate whether transgenic overexpression of the zebrafish BAFF leads to immunoglobulin changes and/or early maturing of the immune system, a Tol2-GFP-2A-BAFF/His recombinant plasmid was constructed by inserting a 2A peptide between the green fluorescent protein (GFP) and BAFF sequences. Functional GFP and BAFF proteins were expressed separately and confirmed in HeLa cells. The relative expression of immune-related genes (IgLC-1, IgLC-2, IgLC-3, IgD, IgM and IL-4), early lymphoid markers (Ikaros, Rag-1 and TCRAC), and the protooncogene Bcl-2 were evaluated by quantitative polymerase chain reaction (PCR) in F0 founder of transgenic zebrafish juveniles and adults. Ectopic expression of BAFF in adults was confirmed using Western blots and was shown to upregulate IgLC-1, IgLC-2, IgD, IgM, IgZ/T, Ikaros, Rag-1, TCRAC, IL-4 and Bcl-2 expression in juveniles on day 21 and IgLC-1, IgLC-2, IgD, IgM, IgZ/T, Rag-1, TCRAC and Bcl-2 expression in zebrafish three months postfertilization. The relative titers of specific IgM against Edwardsiella tarda WED were assessed using modified enzyme-linked immunosorbent assay (ELISA) with the whole body homogenate of zebrafish and demonstrated a significant increase in BAFF-transgenic group. Therefore, our findings provided novel insight into further exploration of modulating adaptive immunity and studying autoimmune diseases caused by regulating BAFF.     

利用Tol2转座子构建斑马鱼心脏组织特异表达转基因载体及其表达分析

为了制备用于在斑马鱼心脏中特异表达目的基因的转基因载体,通过分子克隆的方法对能够在斑马鱼心脏中特异表达EGFP报告基因的Tol2载体进行了改造,在原有的CMLC2启动子与EGFP编码区之间插入带有多克隆位点的IRES序列,获得pTol2-CMLC2-IRES-EGFP转基因表达载体,该载体可以实现在同一个启动子CMLC2的驱动下分别同时表达目的基因和EGFP;为了验证该表达载体的有效性,进一步在CMLC2启动子与IRES序列之间插入DsRed-Monome编码区,利用得到的pTol2-CMLC2-RED-IRES-EGFP转基因载体显微注射到斑马鱼单细胞期胚胎中进行表达分析,结果表明外源目的基因DsRed-Monome和报告基因EGFP均能以相同的表达模式在斑马鱼心脏组织中特异表达。pTol2-CMLC2-IRES-EGFP转基因表达载体的成功构建对于建立心脏发育候选基因的斑马鱼转基因实验模型具有重要意义。     

Recombinant Tol2 transposase with activity in Xenopus embryos

The Tol2 transposon system is a useful gene transduction technique, but the injection of mRNA is not sufficiently effective in Xenopus embryos to express Tol2 transposase (Tol2TP). To overcome this, we bacterially synthesized recombinant Tol2TP (rTol2TP) protein and showed that rTol2TP efficiently excised the Tol2 element from an injected donor plasmid in Xenopus embryos. Furthermore, injected embryos exhibited uniform and ubiquitous expression of an EGFP reporter gene placed within the Tol2 element. Importantly, size-exclusion chromatography suggests that rTol2TP forms a tetramer, which differs from the reported hexamer formed by Hermes transposase, although both belong to the same hAT family. The use of rTol2TP may facilitate efficient gene transduction in Xenopus, and the biochemical characterization of Tol2TP.     

Efficient transient rescue of hematopoietic mutant phenotypes in zebrafish using Tol2‐mediated transgenesis

Phenotypic rescue experiments have been commonly used in zebrafish since it is convenient to study the causality of mutant phenotypes just by injecting mRNA into embryos. However, this strategy is only effective for phenotypes at early embryonic stages due to mRNA instability. For later developmental stages, DNA constructs are used to express exogenous genes, while it is usually ineffective owing to the problem of mosaicism. This study attempted to solve the problem by using Tol2‐mediated transgenesis. As a model case, we used vlad tepes (vlt), a zebrafish gata1 mutant, whose phenotypes have never been able to be rescued at later stages by transient rescue experiments. Blood cell‐specific transgenic expression of gata1 was driven by its own promoter/enhancer elements. The co‐injection of a Tol2‐donor plasmid containing gata1 cDNA and transposase mRNA efficiently rescued the bloodless phenotypes of vlt even in day 12 larvae when definitive erythropoiesis took place with primitive erythropoiesis. This Tol2‐mediated rescue is therefore considered to be a quick and easy method for analyzing the mutant phenotypes in zebrafish.     

Efficient Transfection Strategy for the Spatiotemporal Control of Gene Expression in Zebrafish

Functional analyses of gene function by knockdown and expression approaches strongly enhance the genetic study of development. In vivo application of the introduction of inhibitors of gene expression, mRNA, and expression constructs in the target region make it possible to perform region- and stage-specific regulation of gene function in a simple manner. As a basic tool for the conditional regulation of gene expression in target tissue, we present methods for the efficient introduction of antisense morpholino oligonucleotide (MO), mRNA, and expression plasmid constructs into early and later stage zebrafish embryo and larva. Lipofection of a neuron-specific expression construct plasmid encoding green fluorescent protein (GFP) into optic vesicle resulted in clear GFP expression in the retinotectal pathway in hatched larva. Co-lipofection of MO and GFP mRNA to the presumptive head region resulted in brain-specific knockdown of the gene in mid-stage embryos.     

Highly-restricted, cell-specific expression of the simian CMV-IE promoter in transgenic zebrafish with age and after heat shock

Promoters with high levels of ubiquitous expression are of significant utility in the production of transgenic animals and cell lines. One such promoter is derived from the human cytomegalovirus immediate early (CMV-IE) gene. We sought to ascertain if the simian CMV-IE promoter (sCMV), used extensively in non-mammalian vertebrate research, also directs intense, widespread expression when stably introduced into zebrafish. Analysis of sCMV-driven expression revealed a temporal and spatial pattern not predicted by studies using the hCMV promoter in other transgenic animals or by observations of early F0 embryos expressing injected sCMV-reporter plasmids. Unexpectedly, in transgenic fish produced by both integration of linearized plasmid or Tol2-mediated transgenesis, sCMV promoter expression was generally observed in a small population of cells in telencephalon and spinal cord between days 2 and 7, and was thereafter confined to discrete regions of CNS that included the olfactory bulb, retina, cerebellum, spinal cord, and lateral line. In skeletal muscle, intense transgene expression was not observed until well into adulthood (>2-3 months post-fertilization). One final unexpected characteristic of the sCMV promoter in stable transgenic fish was tissue-specific responsiveness of the promoter to heat shock at both embryonic and adult stages. These data suggest that, in the context of stable transgenesis, the simian CMV-IE gene promoter responds differently to intracellular regulatory forces than other characterized CMV promoters.