PB-GF转座子系统的构建及其在真核细胞中转座的研究

目的构建能在真核细胞中发生转座的PB-GF(piggyBac gene-finder)转座子系统,作为发现和研究编码基因的工具。方法通过克隆技术,构建PB转座子载体pPB[CMV-neo]和PB转座酶表达载体pCAG-PBase,转染HeLa细胞,G418筛选和亚甲基蓝染色,验证PB转座酶在真核细胞中的转座能力。构建PB转座子载体pPB[β-Gal-pA/act-EGFP],使其包含两侧翼端加入PB转座酶识别位点反向末端重复序列(ITR),ITR1和ITR2,以及启动子陷阱和PolyA陷阱的DNA片段;同时与PB转座酶表达载体pCAG-PBase一起,构成PB-GF转座子系统。将PB-GF转座子系统转染HeLa细胞,通过荧光显微镜和流式细胞仪检测PolyA陷阱报告基因EGFP的表达;通过X-gal染色,观察启动子陷阱报告基因β-Gal的表达,明确PB-GF转座酶系统在真核细胞中的转座情况。结果成功构建成以转座子PiggyBac为基础的,含有启动子和PolyA陷阱的PB-GF转座子系统,并用该系统转染HeLa细胞24 h后观察到有相当数量的EGFP和LacZ阳性的细胞出现,转座效率高达26%。结论成功构建PB-GF转座子系统并可以在真核细胞中发生转座。     

piggyBac转座子调节pPiggyBac-Rb质粒的构建*○

背景：外源性Rb基因的导入是抑制视网膜母细胞瘤细胞增殖的有效途径,piggyBac转座子介导的外源基因表达载体高效安全,在基因治疗方面具有广阔的应用前景。 目的：探讨piggyBac转座子介导的外源性Rb基因的转染效率及其对视网膜母细胞瘤细胞的抑制作用。 方法：采用RT-PCR技术扩增Rb基因编码区Rb cDNA,定向插入到载体pPiggyBac,获得PiggyBac调节的Rb表达质粒pPiggyBac-Rb;通过单独转染或合并pPiggyBac-helper转染人视网膜母细胞瘤细胞SO-RB50。 结果与结论：考马斯亮蓝染色证明piggyBac转座子系统的转染效率最高,荧光定量PCR以及免疫荧光实验证明其介导的目的基因Rb与宿主SO-RB50细胞基因组整合效率最高且可在细胞中长期稳定表达,MTT实验证明经其所介导的外源性Rb基因表达对细胞活性影响最为显著。结果提示piggyBac转座子有可能成为视网膜母细胞瘤基因治疗的安全有效载体。关键词：piggyBac转座子;SO-RB50细胞;转染;Rb基因;基因治疗 doi:10.3969/j.issn.1673-8225.2012.20.033     

AR-GFP融合基因真核表达载体的构建及在HEK293细胞中的表达

目的：构建由绿色荧光蛋白（GFP）标记的人醛糖还原酶（AR）融合基因真核表达载体并在HEK293细胞中表达。 方法:应用DNA重组技术构建AR与GFP的融合基因（AR-GFP），将AR-GFP插入pcDNA3.1/myc-HisA真核表达载体中，通过磷酸钙共沉淀转染HEK293细胞，倒置荧光显微镜评价转染效率，Western blotting 技术检测AR基因在转染细胞中的整合及表达，分光光度法（UV法）测定AR表达活性，用公认的AR抑制剂（ARI）反证活性AR的存在。结果:荧光显微镜对转染细胞的观察及Western blotting 结果证实AR-GFP融合蛋白在HEK293细胞中表达，UV法测活、ARI抑制试验均未证实其生物学活性。 结论:转染的HEK293细胞可成功地表达AR-GFP融合蛋白，但不能成为ARI真核细胞筛选模型。     

过表达Sprouty2分子促进HEK293T细胞增殖与存活

目的构建表达人Sprouty 2(SPRY2)基因的重组表达载体pDC315/hSPRY2,转染人胚肾(HEK)293T细胞表达目的蛋白,初步探讨其对HEK293T细胞增殖与存活的影响。方法构建hSPRY2重组腺病毒表达载体,体外转染HEK293T细胞,以Western blot法检测目的蛋白的表达,以CCK-8法检测SPRY2对HEK293T细胞增殖或去血清条件下细胞存活的影响。结果成功构建重组表达载体pDC315/hSPRY2,在转染的HEK293T细胞中检测到目的蛋白hSPRY2的表达,转染pDC315/hSPRY2组HEK293T细胞的增殖率及存活率均明显高于对照组即转染pDC315/EGFP组(P0.05)。结论成功构建了hSPRY2重组表达载体,过表达hSPRY2可促进HEK293T细胞的增殖与存活。     

过表达Sprouty2分子促进HEK293T细胞增殖与存活北大核心CSCD

目的构建表达人Sprouty 2(SPRY2)基因的重组表达载体pDC315/hSPRY2,转染人胚肾(HEK)293T细胞表达目的蛋白,初步探讨其对HEK293T细胞增殖与存活的影响。方法构建hSPRY2重组腺病毒表达载体,体外转染HEK293T细胞,以Western blot法检测目的蛋白的表达,以CCK-8法检测SPRY2对HEK293T细胞增殖或去血清条件下细胞存活的影响。结果成功构建重组表达载体pDC315/hSPRY2,在转染的HEK293T细胞中检测到目的蛋白hSPRY2的表达,转染pDC315/hSPRY2组HEK293T细胞的增殖率及存活率均明显高于对照组即转染pDC315/EGFP组(P<0.05)。结论成功构建了hSPRY2重组表达载体,过表达hSPRY2可促进HEK293T细胞的增殖与存活。     

可溶性CD20在HEK293T细胞中的高效表达

目的构建CD20真核表达载体,在HEK293T细胞中高效表达CD20蛋白。方法以人外周血单个核细胞(PBMC总RNA为模板,反转录PCR扩增得到CD20基因,并插入真核表达载体p CMV3-C-His,PCR初步验证后,再测序验证。将重组质粒瞬时转染HEK293T细胞进行蛋白表达,ELISA、Western blot法检测CD20蛋白表达,优化表达条件。结果 PCR与测序均显示成功插入CD20基因。转染HEK293T细胞72 h后,Western blot法检测到细胞内和细胞培养上清液中的CD20蛋白。HEK293T细胞传代次数与细胞培养上清液中CD20表达量相关。结论成功构建CD20真核表达载体,高效表达CD20可溶性蛋白。     

ureI和ctB-ureI真核质粒构建及表达分析

目的:构建ureI和ctB-ureI真核表达质粒并分析其在细胞的表达情况。方法:用pIRES-RD2真核表达载体分别在5′端连接ureI和ctB-ureI基因,经双酶切和测序鉴定正确后,相应质粒用脂质体转染HEK293T细胞,用荧光显微镜观察荧光标签,确定质粒的转染率;用人抗幽门螺旋杆菌抗体和马抗CtB抗体,采用免疫荧光和免疫酶组织化学等方法研究该两种真核表达质粒在细胞中目的蛋白表达情况。结果:成功构建了ureI和ctB-ureI的真核表达质粒pIRES-RD2-ureI和pIRES-RD2-ctB-ureI,用此质粒转染HEK293T细胞48～72h后,荧光显微镜显示该两种质粒的转染率约为80%;用免疫荧光和免疫酶组织化学方法表明了该两种基因均能在HEK293T细胞表达并有免疫反应性,表达的ureI主要分布在胞质内或细胞膜附近,ctB-ureI在CtB信号肽引导下分布于HEK293T细胞质、细胞膜附近以及细胞外。结论:成功构建了真核载体pIRES2-DsRed2-ureI和pIRES2-DsRed2-ctB-ureI,目的蛋白表达于HEK293T细胞胞质内并有一定免疫反应性。     

建立人促甲状腺素受体α亚基HEK 293T细胞稳定表达株

为研究抗甲状腺多肽的特异性与亲和力,本研究建立了人促甲状腺素受体(TSHR)α亚基(hTSHRA)稳定表达株。构建hTSHRA到慢病毒质粒GV218上形成GV218-hTSHRA,挑取GV218-hTSHRA构建物阳性大肠杆菌克隆,提取DNA测序结果与人TSHRA序列比对,100%吻合。结果显示:GV218-hTSHRA转染到HEK 293T后,Western blot显示,HEK 293T细胞提取蛋白出现52kD的hTSHRA目的条带。GV218-hTSHRA、辅助质粒在HEK 293T细胞中包装后,HEK 293T细胞表达绿色荧光蛋白。收取浓缩的包装病毒,qPCR测定滴度达到了2×108 TU/mL。再将包装病毒转染到HEK 293T细胞中,hTSHRA即表达在HEK 293T细胞上,细胞传代后hTSHRA仍然稳定表达。结果表明获得GV218-hTSHRA包装质粒,并建立了hTSHRA-HEK 293T稳定表达株,为研究抗甲状腺多肽与人TSHRα亚基特异性与亲和力提供了必要的实验材料。     

Salusin-a基因真核表达载体的构建及在HEK293细胞中的表达

目的:研究增强型绿色荧光蛋白真核表达载体Salusin-a(pEG-FP-Salusin-a)的构建及在人胚胎肾细胞系293细胞(HEK293)中的表达。方法:提取人单核细胞系THP-1细胞Salusin-a的mRNA,逆转录多聚酶链反应(RT-PCR)扩增Salusin-a基因,克隆入pEGFP-N3载体,双酶切、菌落PCR及测序鉴定pEGFP-Salusin-a质粒。用脂质体转染pEGFP-Salusin-a入HEK293细胞,分为转染细胞组、质粒对照组和空白对照组,检测分析各组荧光蛋白和Salusin-a基因的表达。结果:成功构建pEGFP-Salusin-a质粒,并转染HEK293,细胞转染效率86%,转染细胞组和质粒对照组绿色荧光蛋白的表达明显高于空白对照组(P0.05);转染细胞组Salusin-amRNA的表达明显高于质粒对照组和空白对照组(P0.05)。结论:重组pEGFP-Salusin-a质粒能转染HEK293细胞并表达Salusin-a,为Salusin-a基因功能的进一步研究提供了实验基础。     

慢病毒载体介导下DJ-1过表达细胞模型的建立

目的构建人野生型DJ-1及其L166P突变体的慢病毒载体并探讨慢病毒载体在构建基因过表达细胞模型中的作用。方法分别构建野生型DJ-1与L166P突变型DJ-1慢病毒载体质粒。进行测序确定比对正确后,进行质粒的大量扩增与制备并转染包装细胞系HEK293T细胞,荧光法和Western blot检测野生型DJ-1与L166P突变型DJ-1在细胞系中的表达。在确定目的蛋白正确表达之后,大量转染HEK293T细胞进行包装并生产携带目的基因的慢病毒颗粒。测定病毒上清滴度后感染PC12细胞,荧光显微镜和Western blot观察GFP荧光强度以及目的蛋白的表达,确定病毒的感染效率。结果成功构建携带DJ-1野生型及其突变体的慢病毒载体。该病毒载体可以转染进入HEK293T细胞内且目的蛋白能够正确表达。LV-DJ-1与LV-DJ-1/L166P的病毒滴度分别为2×10~9TU/m L与2×10~8TU/m L。病毒上清可以高效感染PC12细胞,绝大多数细胞可表达目的蛋白。外源野生型DJ-1和L166P突变体的蛋白表达量分别是内源性含量的315%和285%。结论慢病毒感染细胞效率很高,是很好的制备基因过表达细胞的方法。通过慢病毒载体介导,本研究获得了DJ-1及其突变体的过表达细胞模型。该模型可以用于后续DJ-1功能研究。     

Germline transgenesis of zebrafish using the medaka Tol1 transposon system

Tol1 is a DNA-based transposable element first identified from an albino mutant medaka fish. It has been demonstrated to function as an efficient gene transfer vector in mammalian cells. We now demonstrate Tol1 germline transgenesis in zebrafish. A construct containing the green fluorescence protein (GFP) reporter gene inserted between the Tol1 arms was microinjected together with Tol1 transposase mRNA into fertilized eggs. Sustained GFP expression was observed in 88% of 1-month-old fish, suggesting efficient transposon integration into somatic cells. Eleven of 24 adult GFP-positive fish yielded GFP-positive progeny. Sequencing analysis of Tol1 insertion sites in GFP-positive progeny confirmed Tol1 transposition-mediated integrations into zebrafish chromosomes. We also observed functional independence of the Tol1 transposase-substrate system from that of Tol2, another medaka-derived transposon. Coupled with its previously demonstrated maximal cargo capacity of >20 kb, Tol1 could serve as a useful addition to the zebrafish genetic engineering toolbox.     

Tol2 transposon-mediated enhancer trap to identify developmentally regulated zebrafish genes in vivo.

We have used the Tol2 transposable element to design and perform effective enhancer trapping in zebrafish. Modified transposon DNA and transposase RNA were delivered into zebrafish embryos by microinjection to produce heritable insertions in the zebrafish genome. The enhancer trap construct carries the EGFP gene controlled by a partial epithelial promoter from the keratin8 gene. Enhanced green fluorescent protein (EGFP) is used as a marker to select F1 transgenic fish and as a reporter to trap enhancers. We have isolated 28 transgenic lines that were derived from the 37 GFP-positive F0 founders and displayed various specific EGFP expression patterns in addition to basal expression from the modified keratin 8 promoter. Analyses of expression by whole-mount RNA in situ hybridization demonstrated that these patterns could recapitulate the expression of the tagged genes to a variable extent and, therefore, confirmed that our construct worked effectively as an enhancer trap. Transgenic offspring from the 37 F0 EGFP-positive founders have been genetically analyzed up to the F2 generation. Flanking sequences from 65 separate transposon insertion sites were identified by thermal asymmetric interlaced polymerase chain reaction. Injection of the transposase RNA into transgenic embryos induced remobilization of genomic Tol2 copies producing novel insertions including some in the germ line. The approach has great potential for developmental and anatomical studies of teleosts.     

骨髓间充质干细胞中突变型人低氧诱导因子1α真核表达载体的表达

背景：转基因小鼠体内实验证实,重组的低氧诱导因子1α可以促进皮肤形态功能正常的血管新生。 目的：构建能够在常氧条件下同时表达突变型低氧诱导因子1α目的蛋白和绿色荧光蛋白报告分子的新型腺病毒真核表达载体,并转染SD大鼠骨髓间充质干细胞,检测该基因在细胞中的表达情况。 方法：利用Lipofectamine 2000介导将构建成功的重组腺病毒真核表达载体pAd-HIF1αmu- IRES-hrGFP-1转染HEK293A细胞,包装病毒,以最佳感染指数=50将重组腺病毒转染大鼠骨髓间充质干细胞,并设3个对照组,即阳性对照组：转染Ad-CMV-HIF1α-IRES-hrGFP-1;阴性对照组：转染Ad-CMV-IRES-hrGFP-1组;空白组：未转染病毒。 结果与结论：①腺病毒载体成功转染HEK293A细胞,包装成功,细胞内有大量绿色荧光表达。②转染突变型低氧诱导因子1α腺病毒表达载体的细胞在常氧条件下蛋白表达量明显高于转其他3组,3个对照组间差异无显著性意义(P > 0.05)。提示突变型腺病毒真核表达载体Ad-HIF1α-IRES-hrGFP-1在HEK293A内成功包装;突变后低氧诱导因子1α基因能够在常氧条件下大量且高效表达。     

pcDNA3.1+-MBL 真核表达载体构建及其在不同哺乳动物细胞株中的表达

目的　比较人甘露聚糖结合凝集素 (MBL)基因在不同哺乳细胞株中的表达效率。方法　应用PCR从含有中国人野生型MBLcDNA的质粒pGEM MBL中扩增目的基因片段 ,将其克隆至pcDNA3.1 表达载体 ,经酶切及测序鉴定后 ,以电穿孔法转染CHO、HEPG2和HEK2 93细胞 ,以RT PCR、ELISA分析其在 3株细胞中的表达情况。结果　从pGEM MBL中扩增得到约 75 0bp的MBLDNA片段 ,构建成重组表达载体pcDNA3.1 MBL ,经酶切出现 75 0bp片段 ,测序鉴定与预期的完全一致。将其转染进细胞后 ,经G4 18选择转染子并克隆化培养 ,RT PCR和ELISA分析显示 ,在CHO和HEK2 93细胞中及培养上清液中分别有较高的MBLmRNA和蛋白表达 ,而HEPG2细胞表达较低。结论　人MBL在CHO和HEK2 93细胞中的表达效率较高 ,为今后在哺乳动物细胞中高效表达人MBL积累了经验     

The Tol2kit: a multisite gateway-based construction kit for Tol2 transposon transgenesis constructs.

Transgenesis is an important tool for assessing gene function. In zebrafish, transgenesis has suffered from three problems: the labor of building complex expression constructs using conventional subcloning; low transgenesis efficiency, leading to mosaicism in transient transgenics and infrequent germline incorporation; and difficulty in identifying germline integrations unless using a fluorescent marker transgene. The Tol2kit system uses site-specific recombination-based cloning (multisite Gateway technology) to allow quick, modular assembly of [promoter]-[coding sequence]-[3' tag] constructs in a Tol2 transposon backbone. It includes a destination vector with a cmlc2:EGFP (enhanced green fluorescent protein) transgenesis marker and a variety of widely useful entry clones, including hsp70 and beta-actin promoters; cytoplasmic, nuclear, and membrane-localized fluorescent proteins; and internal ribosome entry sequence-driven EGFP cassettes for bicistronic expression. The Tol2kit greatly facilitates zebrafish transgenesis, simplifies the sharing of clones, and enables large-scale projects testing the functions of libraries of regulatory or coding sequences.     

小鼠精子发生相关蛋白3基因在小鼠生精细胞的特异表达及对HEK 293T细胞凋亡和自噬的影响

目的检测小鼠精子发生相关蛋白3(spata3)基因在小鼠生精细胞的表达情况,并借助过表达细胞模型进一步分析该基因对人胚肾HEK 293T细胞凋亡及自噬的影响,旨在探讨spata3在精子发生过程中的意义。方法分别采用RT-PCR和免疫印迹法检测spata3基因mRNA及蛋白产物在小鼠各组织中的表达;应用免疫组织化学和免疫荧光染色观察SPATA3蛋白在生精细胞中的定位;借助脂质体将真核表达载体Plv-EGFP-2(a)purospata3瞬时转染HEK 293T细胞,进一步在蛋白水平分析细胞凋亡相关蛋白Caspase-3、多聚ADP-核糖聚合酶(PARP)、BAX和Bcl-2及自噬相关蛋白LC3A/B的变化。结果 Spata3基因及其编码产物在小鼠睾丸组织特异表达;粗线期精母细胞和圆形精子细胞的胞质与胞核均有显著SPATA3蛋白的阳性着色,长形精子细胞的胞质也有大量分布;过表达spata3的HEK 293T细胞内活化型Caspase-3和PARP降解产物的含量较对照组差异无显著性,BAX表达量0.815±0.020较裸细胞组0.469±0.012和空载体转染组0.588±0.018均有所增高,Bcl-2含量0.214±0.020低于裸细胞0.507±0.021和空载体转染组0.545±0.024,LC3A/B-Ⅱ的表达量0.741±0.037则显著高于裸细胞组0.136±0.011和空载体转染组0.169±0.012。结论 Spata3基因在小鼠生精细胞特异表达,过表达spata3对HEK 293T细胞凋亡无明显影响,但可以促进细胞自噬。     

酪氨酸酶基因在HEK293细胞表达的MRI评价

目的:以酪氨酸酶基因作为报告基因转染HEK293细胞, 利用其合成大量黑色素而能被MRI检测的特性来反映基因表达的情况, 以探索磁共振成像(MRI)评价体外细胞基因表达的方法。方法:以脂质体将含酪氨酸酶基因完全cDNA的pcDNA3tyr质粒转染到HEK293细胞, 以MRIT₁WI、T₁WI/SPIR、T₂WI序列扫描转染细胞, 观察表达的黑色素的MRI信号。应用Fontana染色检测黑色素的合成, RT-PCR检测酪氨酸酶基因的cDNA片段, 以进一步验证酪氨酸酶基因的转染与表达。结果:(1)pcDNA3tyr质粒转染进入HEK293细胞并在其中表达生成黑色素, 转染5μg、10μg、20μg质粒的10⁶个细胞内生成的黑色素能够被MRI检测到并在MRIT₁WI、T₁WI/SPIR、T₂WI检查呈高信号, MRI信号强度与转染质粒量成正相关。(2)Fontana染色法检测到HEK293细胞内的黑色素颗粒;(3)采用RT-PCR方法检测到转染的HEK293细胞含酪氨酸酶基因的cDNA片段。结论:MRI能够检测到HEK293细胞内由外源基因表达合成的黑色素, 说明影像学与分子生物学技术结合可以评价体外细胞基因表达的情况。     

TCAB1基因RNAi慢病毒载体的构建及在人子宫内膜间质细胞中沉默TCAB1基因的效应

目的构建TCAB1基因RNAi慢病毒载体,为子宫内膜异位症(EMs)的基因靶向治疗提供理论依据。方法化学合成3条靶向TCAB1基因的siRNA,转染HEK-293T细胞,24h后采用荧光素酶报告系统筛选有效siRNA,其对TCAB1mRNA有明显抑制作用。针对已经筛选确定的TCAB1基因RNAi有效靶序列,合成靶序列的Oligo DNA,退火形成双链DNA,与经XhoI和HpaI酶切后的LV1载体[含U6启动子和绿色荧光蛋白(GFP)]连接产生LV1-shRNA慢病毒载体,PCR筛选阳性克隆,测序鉴定。用LV1-shRNA、pRev、pVsvg、pcgv四质粒共转染包装细胞HEK-293T细胞,包装产生慢病毒,分别于转染后48h、72h收取病毒上清,感染人子宫内膜间质细胞(ESC),进行RT-qPCR检测TCAB1基因相对表达量,检测LV1-shRNA干扰效率。结果 PCR和测序证实,成功构建TCAB1 shRNA的慢病毒载体LV1-shRNA。结论成功构建TCAB1基因的RNAi慢病毒载体,其转染ESC细胞后显著抑制了TCAB1的表达,为子宫内膜异位症的基因靶向治疗提供了实验基础。     

Transposon-mediated gene transfer into adult and induced pluripotent stem cells

Transposon technology is a particularly attractive non-viral gene delivery paradigm that allows for efficient genomic integration into a variety of different cell types. In particular, transposon-mediated gene transfer is a promising tool for stem cell research, by virtue of its ability to efficiently and stably transfer genes into adult and induced pluripotent stem (iPS) cells. Moreover, transposons open up new perspectives for non-viral-mediated stem cell-based gene therapy. Several transposon systems, especially the Sleeping Beauty (SB), the piggyBac (PB) and Tol2, have been optimized for gene transfer into mammalian cells. In particular, SB resulted in stable gene transfer into various adult stem cells including human CD34(+) hematopoietic stem cells (HSCs), myoblasts and mesenchymal stem cells (MSCs). This has been confirmed with PB, yielding stable gene transfer in human CD34(+) HSCs. Recently, PB transposons were used to deliver the genes encoding the reprogramming factors into somatic cells making it an attractive technology for generating iPS cells. Subsequent de novo expression of the PB transposase resulted in traceless excision of the reprogramming cassette. This prevented inadvertent re-expression of the reprogramming factors obviating some of the concerns associated with the use of integrating vectors. Transposons have also been used as a novel non-viral paradigm to coax differentiation of iPS cells into their desired target cells by forced expression of specific differentiation factors. This review focuses on the emerging potential of transposons for gene transfer into stem cells and its implications for gene therapy and regenerative medicine.