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。     

利用流式细胞仪分选拟南芥根尖发育早期非根毛细胞

建立了应用流式细胞仪分选植物特定类型细胞的方法。以拟南芥(Arabidopsis thaliana)Wer::GFP转基因株系为材料,用激光共聚焦显微镜鉴定GFP的表达位置,采用酶解法制备拟南芥根尖原生质体,应用流式细胞仪荧光激活细胞分选技术(FACS)分选收集GFP阳性细胞,并提取细胞的RNA。结果表明,Wer::GFP转基因株系仅在根表皮发育早期的非根毛细胞中表达GFP;利用酶解法制备的根尖原生质体数目较多;从FACS分选收集的细胞中提取的RNA质量较好,可用于研究特定类型细胞的基因表达谱。应用流式细胞仪分选拟南芥非根毛细胞的方法为研究植物特定类型细胞的基因表达谱及基因功能奠定了技术基础。     

利用流式细胞仪分选拟南芥根尖发育早期非根毛细胞

建立了应用流式细胞仪分选植物特定类型细胞的方法。以拟南芥（Arabidopsis thaliana）Wer：：GFP转基因株系为材料,用激光共聚焦显微镜鉴定GFP的表达位置,采用酶解法制备拟南芥根尖原生质体,应用流式细胞仪荧光激活细胞分选技术（FACS）分选收集GFP阳性细胞,并提取细胞的RNA。结果表明,Wer：：GFP转基因株系仅在根表皮发育早期的非根毛细胞中表达GFP;利用酶解法制备的根尖原生质体数目较多;从FACS分选收集的细胞中提取的RNA质量较好,可用于研究特定类型细胞的基因表达谱。应用流式细胞仪分选拟南芥非根毛细胞的方法为研究植物特定类型细胞的基因表达谱及基因功能奠定了技术基础。     

一种在紫外敏感型视锥细胞中特异表达tdTomato的转基因斑马鱼系的构建

目的鉴于目前对斑马鱼视锥细胞视蛋白运输机制并不明确,且缺乏相应的抗体,本实验拟构建一种可以在视锥细胞中特异表达荧光的转基因斑马鱼。方法利用编码紫外敏感型视蛋白基因(sws1)的启动子,构建了一种在紫外敏感型视锥细胞中特异表达红色荧光蛋白td Tomato的转基因斑马鱼。同时,将td Tomato荧光蛋白与非洲爪蟾rhodopsin蛋白末端44个氨基酸相融合,将该融合蛋白定位于紫外敏感型视锥细胞的外节段,进而可模拟内源性视蛋白的定位。结果共筛选出三种不同品系的转基因斑马鱼,经过免疫组化分析,确定其中一种转基因品系是正确的目标品系。结论该转基因斑马鱼的构建将会为进一步研究视锥细胞中视蛋白的运输机制提供帮助。     

生长激素促进斑马鱼尾鳍的再生

目的建立生长激素过表达的转基因斑马鱼,研究生长激素在斑马鱼尾鳍再生过程中的作用。方法利用Gateway技术构建表达质粒"pDestTol2CG2; ubi:GH-polyA",在一细胞期显微注射表达质粒和转座酶mRNA后,通过荧光显微镜和qPCR技术筛选鉴定GH过表达的转基因斑马鱼。将斑马鱼分为对照组(野生型)和生长激素过表达组,尾鳍横切后,记录分析斑马鱼尾鳍再生过程。结果转基因斑马鱼中心脏被绿色荧光蛋白标记。荧光定量PCR检测结果显示GH表达水平显著高于对照组(P<0.05)。斑马鱼尾鳍横断后,生长激素过表达组的再生速度显著提高(P<0.05)。结论建立稳定生长激素过表达的转基因斑马鱼品系,过表达生长激素能够提高斑马鱼尾鳍再生速度。     

绿色荧光转基因大鼠模型的建立

目的随着干细胞研究的推进,大鼠干细胞的研究日趋迫切。本研究旨在为活体荧光影像系统、干细胞归巢、细胞移植体内示踪研究,提供绿色荧光蛋白EGFP转基因大鼠模型。方法通过显微注射方式获得EGFP转基因大鼠,采用活体荧光影像系统、激光共聚焦显微镜,对EGFP转基因大鼠各个组织的荧光表达水平进行比较;采用流式细胞术检测转基因大鼠血液和骨髓细胞、骨髓干细胞的荧光标记率,筛选骨髓干细胞高效标记绿色荧光的转基因大鼠。结果建立了心脏、肝脏、肌肉、肺、胰腺、脑、膀胱、胃、肾脏、肠和脾脏组织中,系统性表达EGFP的SD-TgN（ACT-EGFP-1）ZLFILAS转基因大鼠;流式细胞术检测表明,该品系血液细胞绿色荧光标记率为94.4%,骨髓干细胞绿色荧光标记率为97.8%。结论建立了多组织系统性高表达绿色荧光,骨髓干细胞荧光标记率高达95%以上的转基因大鼠,为影像分析,造血干细胞的归巢等研究提供了大鼠模型。     

用western blotting方法检测乙醇固定细胞中细胞周期素的表达

用western-blotting法测定乙醇固定细胞中细胞周期素（cyclin)的表达情况,探索western-blotting和流式细胞仪对固定细胞在流式细胞术分选前或分选后对同批标本进行蛋白同步分析的可行性,采用对数生长期的Molt-4细胞,经两种常规固定方法固定后,用western-blotting方法检测cyclinA,B1、D和E的表达,另取乙醇固定经流式细胞仪分选的G1期和G2/M期细胞裂解后,用western-blotting方法检测不同时相细胞中cyclinB1的表达。结果显示从固定细胞中提取的蛋白经western-blotting检测可得到清晰且分子量正确的条带,两种不同方法固定的细胞中检测到的cyclins表达未见明显差异。乙醇固定细胞经分选后提取蛋白行western-blotting检测可见G2/M期细胞的cyclinB1有明显表达而G1期细胞不明显。细胞进行固定,洗涤,染色和分选等处理后不影响western-blotting对其cyclins表达的分析。说明用western-blotting和流式细胞仪对固定细胞在流式细胞术分选前或分选后进行蛋白同步分析是可行的。     

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...     

利用慢病毒载体快速建立表达外源基因的哺乳动物细胞系

建立稳定表达外源基因的哺乳动物细胞系是一项重要的生物技术,介绍了联合使用慢病毒载体和流式细胞仪分选技术来建立稳定表达绿色荧光蛋白的293T细胞系.结果表明,在1个月左右的时间里即可获得表达绿色荧光蛋白的293T细胞系,阳性细胞率高达96.5％,而且建立的细胞系能够稳定传代.因此,联合慢病毒载体和流式细胞仪分选技术的策略对于建立稳定表达外源基因的哺乳动物细胞系快捷和可靠.     

快速筛选高效表达重组蛋白毕赤酵母菌株新方法的建立及评价

毕赤酵母是当前应用最为广泛的重组蛋白表达系统之一,文中建立了一种快速筛选高效表达重组蛋白的毕赤酵母菌株的新方法。首先,对内质网转膜蛋白Sec63融合表达增强型绿色荧光蛋白EGFP的改造菌株GS115-E表达重组蛋白的能力进行检测;之后将携带不同拷贝数的植酸酶phy基因或木聚糖酶xyn基因的质粒转化进入GS115-E中,得到具有不同植酸酶或木聚糖酶表达水平的重组菌株,分别检测不同菌株的EGFP与重组蛋白的表达水平;最后,利用分选型流式细胞仪,根据绿色荧光值的高低对包含不同植酸酶表达水平的重组菌株的菌群进行分选。结果显示重组菌株中EGFP的荧光值与重组蛋白的活性表达水平之间具有良好的线性相关性(0.8|R|1),且利用流式细胞仪可高效地从混合菌群中筛选得到高产菌株,所分选得到的高荧光菌株在摇瓶发酵120 h时植酸酶表达水平是低荧光菌株的4.09倍。本方法通过检测菌株的EGFP荧光值代替检测重组蛋白的表达水平和活性,从而实现高表达菌株的筛选,大大提高了其应用的便捷性及通用性。与流式细胞仪、液滴微流控等高通量筛选仪器或技术结合将进一步提高筛选的速度与通量,为筛选获得高效表达重组蛋白的毕赤酵母菌株提供了简便、快速的新途径。     

The influence of donor nucleus source on the outcome of zebrafish somatic cell nuclear transfer

The success of nuclear reprogramming following somatic cell nuclear transfer (SCNT) is thought to depend on factors present in the egg. Little is known about the role - if any - played by the somatic cell type on the outcome of the procedure. We tested whether cells of different lineages might have different capacities for reprogramming following SCNT, comparing cells isolated from five different tissues of transgenic zebrafish for their developmental potential when used as SCNT donor cells. We used transgenic zebrafish lines expressing green fluorescence protein under an endogenous tissue-specific promoter: HGn62A-skin, HGn28A-skin, HGn8E-heart, HG21C-fin and notochord and HGn30A-hatch gland. We analyzed the efficiency of cloning, as measured by reconstructed embryos that developed up to the hatched-fry stage. Specifically, donor cells of fin and notochord origin yielded the best rate of cloned fish production. All of the other cell types used were capable of producing cloned fish, albeit with significantly lower efficiency. These results indicate that the type of zebrafish cells used for SCNT can influence the outcome of the procedure. Future epigenetic analysis of these cells will help determine specific chromatin profiles in somatic cells that have an impact on nuclear reprogramming procedures.     

Establishment and characterization of CAG/EGFP transgenic rabbit line

Cell marking is a very important procedure for identifying donor cells after cell and/or organ transplantation in vivo. Transgenic animals expressing marker proteins such as enhanced green fluorescent protein (EGFP) in their tissues are a powerful tool for research in fields of tissue engineering and regenerative medicine. The purpose of this study was to establish transgenic rabbit lines that ubiquitously express EGFP under the control of the cytomegalovirus immediate early enhancer/beta-actin promoter (CAG) to provide a fluorescent transgenic animal as a bioresource. We microinjected the EGFP expression vector into 945 rabbit eggs and 4 independent transgenic candidate pups were obtained. Two of them died before sexual maturation and one was infertile. One transgenic male candidate founder rabbit was obtained and could be bred by artificial insemination. The rabbit transmitted the transgene in a Mendelian manner. Using fluorescence in situ hybridization analysis, we detected the transgene at 7q11 on chromosome 7 as a large centromeric region in two F1 offspring (one female and one male). Eventually, one transgenic line was established. Ubiquitous EGFP florescence was confirmed in all examined organs. There were no gender-related differences in fluorescence. The established CAG/EGFP transgenic rabbit will be an important bioresource and a useful tool for various studies in tissue engineering and regenerative medicine.     

Modified bacterial artificial chromosomes for zebrafish transgenesis

Transgenesis using bacterial artificial chromosomes (BAC) offers greater fidelity in directing desirable expression of foreign genes. Application of this technology in the optically transparent zebrafish with fluorescent protein reporters enables unparalleled visual analysis of regulation of gene expression in a living organism. Here we describe a streamlined procedure of direct selecting multiple BAC clones based on public sequence databases followed by rapid modification with GFP or RFP for transgenic analysis in zebrafish. Experimental procedures for BAC DNA preparation, microinjection of zebrafish embryos and screening of transgenic zebrafish carrying GFP/RFP modified BAC clones are detailed.     

The Fugu tyrp1 promoter directs specific GFP expression in zebrafish: tools to study the RPE and the neural crest‐derived melanophores

In vertebrates, pigment cells account for a small percentage of the total cell population and they intermingle with other cell types. This makes it difficult to isolate them for analyzes of their functions in the context of development. To alleviate such difficulty, we generated two stable transgenic zebrafish lines (pt101 and pt102) that express green fluorescent protein (GFP) in melanophores under the control of the 1 kb Fugu tyrp1 promoter. In pt101, GFP is expressed in both retinal pigment epithelium (RPE) cells and the neural crest‐derived melanophores (NCDM), whereas in pt102, GFP is predominately expressed in the NCDM. Our results indicate that the Fugu tyrp1 promoter can direct transgene expression in a cell‐type‐specific manner in zebrafish. In addition, our findings provide evidence supporting differential regulations of melanin‐synthesizing genes in RPE cells and the NCDM in zebrafish. Utilizing the varying GFP expression levels in these fish, we have isolated melanophores via flow cytometry and revealed the capability of sorting the NCDM from RPE cells as well. Thus, these transgenic lines are useful tools to study melanophores in zebrafish.     

Nitroreductase-mediated cell/tissue ablation in zebrafish: a spatially and temporally controlled ablation method with applications in developmental and regeneration studies

Ablation studies are used to elucidate cell lineage relationships, developmental roles for specific cells during embryogenesis and mechanisms of tissue regeneration. Previous chemical and genetic approaches to directed cell ablation have been hampered by poor specificity, limited efficacy, irreversibility, hypersensitivity to promoter leakiness, restriction to proliferating cells, slow inducibility or complex genetics. Here, we provide a step-by-step protocol for a hybrid chemical-genetic cell ablation method in zebrafish that, by combining spatial and temporal control, is cell-type specific, inducible, reversible, rapid and scaleable. Bacterial Nitroreductase (NTR) is used to catalyze the reduction of the innocuous prodrug metrodinazole (Mtz), thereby producing a cytotoxic product that induces cell death. Based on this principle, NTR is expressed in transgenic zebrafish using a tissue-specific promoter. Subsequent exposure to Mtz by adding it to the media induces cell death exclusively within NTR(+) cells. This approach can be applied to regeneration studies, as removing Mtz by washing permits tissue recovery. Using this protocol, cell ablation can be achieved in 12-72 h, depending on the transgenic line used, and recovery initiates within the following 24 h.     

The Fugu tyrp1 promoter directs specific GFP expression in zebrafish: tools to study the RPE and the neural crest-derived melanophores

In vertebrates, pigment cells account for a small percentage of the total cell population and they intermingle with other cell types. This makes it difficult to isolate them for analyzes of their functions in the context of development. To alleviate such difficulty, we generated two stable transgenic zebrafish lines (pt101 and pt102) that express green fluorescent protein (GFP) in melanophores under the control of the 1 kb Fugu tyrp1 promoter. In pt101, GFP is expressed in both retinal pigment epithelium (RPE) cells and the neural crest-derived melanophores (NCDM), whereas in pt102, GFP is predominately expressed in the NCDM. Our results indicate that the Fugu tyrp1 promoter can direct transgene expression in a cell-type-specific manner in zebrafish. In addition, our findings provide evidence supporting differential regulations of melanin-synthesizing genes in RPE cells and the NCDM in zebrafish. Utilizing the varying GFP expression levels in these fish, we have isolated melanophores via flow cytometry and revealed the capability of sorting the NCDM from RPE cells as well. Thus, these transgenic lines are useful tools to study melanophores in zebrafish.     

基于新的CRISPR/Cas9技术实现斑马鱼LHX9基因的快速编辑及对F0突变体性腺发育的初筛

目的:CRISPR/Cas9系统在斑马鱼的反向遗传学中的到了广泛应用,但突变基因的表型观察往往需要在突变鱼系的F1中进行,费时较长。LHX9作为LIM家族的一种转录因子,在胚胎早期的泌尿生殖嵴中有广泛分布;且LHX9基因敲除的小鼠存在性腺发育不良。本研究拟通过一种新的CRISPR/Cas9基因编辑技术,采用四条sgRNA对LHX9基因进行VASA转基因斑马鱼的基因敲除,以观察该基因缺陷对斑马鱼性腺发育的影响。方法:利用新的CRISPR/Cas9技术,设计四条针对斑马鱼LHX9基因3号外显子的20bp的sgRNA,通过非克隆体外转录得到靶位点的四条sgRNA。将以上靶位点的四条sgRNA与Cas9核酸酶蛋白同时注射入单细胞期的斑马鱼胚胎内,利用PCR鉴定突变型类型和突变比例。通过对LHX9基因突变体的VASA转基因斑马鱼进行荧光观察,发现LHX9基因缺陷的斑马鱼性腺发育的情况。结果:靶向Exon 3的四条sgRNA可成功编辑斑马鱼LHX9基因,敲除效率高达82%,Sanger测序发现产生10种不同的移码突变类型。通过该方法对VASA转基因斑马鱼的LHX9基因进行编辑,发现LHX9基因突变导致dph6的的斑马鱼原始生殖细胞增殖和迁移受到影响。结论:基于4条sgRNA注射的CRISPR/Cas9技术,可以快速地产生具有突变表型的G0斑马鱼,具有应用潜力。LHX9基因敲除导致原始生殖细胞的发育和迁移受到影响,提示该基因参与了斑马鱼早期性腺的发育。