中华按蚊电转化显微注射技术平台的构建及其在基因瞬时过表达中的应用

【目的】构建在中华按蚊 Anopheles sinensis 中的电转化显微注射技术平台,并利用该技术平台实现活体基因瞬时过表达对其进行验证,为开展系统的基因功能研究奠定基础。【方法】以CUY21EDIT Ⅱ电转仪为主体构建中华按蚊中的电转化显微注射技术平台;使用同源重组法构建 EGFP 和 Bm-iAANAT 基因的瞬时过表达质粒,在中华按蚊化蛹3 h时注射该质粒进入蛹体,随即使用电转仪对注射后的蛹进行电击,待注射个体发育至化蛹39 h时,利用体视荧光显微镜观察蛹体表皮着色和发光情况,并通过荧光定量PCR检测 EGFP 和 Bm-iAANAT 的表达。【结果】构建了用于显微注射的 PIZ-modi-Aepub-EGFP-SV 40和 PIZ-modi-Aepub-AANAT- T2A-EGFP-SV 40过表达载体。 PIZ-modi-Aepub-EGFP-SV 40注射组中华按蚊在化蛹39 h时约87.5%的个体成活并正常黑化,这其中约92.7%的个体的表皮检测到明显的绿色荧光,注射无启动子载体 PIZ-modi-EGFP-SV 40并进行电击的阴性对照组和注射过表达载体 PIZ-modi-Aepub-EGFP-SV 40但未进行电击的阳性对照组个体则没有明显的绿色荧光;并且荧光定量PCR结果显示,注射组中发光个体的 EGFP 基因明显高表达。 PIZ-modi-Aepub-AANAT-T 2 A-EGFP-SV 40注射组的蛹在化蛹39 h时有80.4%个体存活,这其中92.2%的个体相对于注射无启动子载体 PIZ-modi-AANAT- T2A -EGFP-SV 40的阴性对照组和注射过表达载体 PIZ-modi-Aepub-AANAT- T2A -EGFP-SV 40但未进行电击的阳性对照组个体黑化明显受阻,且有明显的绿色荧光;并且荧光定量PCR结果显示,报告基因 Bm-iAANAT 和 EGFP 的表达明显提高。【结论】成功构建了在中华按蚊中的电转化显微注射技术平台;通过此技术平台能够便捷、快速和高效地实现报告基因在活蛹中的瞬时过表达,并产生了目的表型。这为中华按蚊功能基因组研究奠定了基础。     

斑马鱼心肌特异性启动子的克隆及其功能鉴定*

目的:探讨心室肌球蛋白重链(vmhc)基因启动子的心肌组织特异性.方法:利用PCR技术从斑马鱼基因组中克隆了vmhc编码区5’上游大小为1952bp的调控区域,应用酶切连接方法将vmhc启动子插入pGEFP-N1质粒,成功构建pEGFP-vmhc重组载体.再应用高保真DNA聚合酶PCR扩增包含vmhc启动子序列,增强型绿色荧光蛋白(EGFP)基因序列及3'UTR序列的基因片段,经过纯化后通过显微注射将vmhc-EGFP基因片段导入斑马鱼受精卵中.结果:注射后的斑马鱼心脏中出现绿色荧光,而其他部位无荧光出现.结论:vmhc启动子能够正确有效地驱动外源基因在斑马鱼心脏中特异表达,适合应用于心血管疾病的基因功能研究,基因靶向治疗等.     

CMV与SP双启动子增强外源基因在小鼠骨骼肌中的表达效率

构建分别含有CMV、肌肉特异启动子SP及双启动子CMV-SP的真核报告基因EGFP表达载体（pCMV-EGFP、pSP-EGFP及pCMV-SP-EGFP）和生长激素释放因子（GRF）表达载体（pCMV-GRF、pSP-GRF和pCMV-SP-GRF）.将3种EGFP表达质粒分别注射至小鼠骨骼肌.注射后1周、2周、3周以及4周时,分别提取肌肉组织RNA,应用半定量RT-PCR检测报告基因（EGFP）的表达量,发现pCMV-SP-EGFP组EGFP表达量显著高于pCMV-EGFP组和pSP-EGFP组（P<0.01）;经荧光检测得到了较强的荧光信号.将3种GRF表达质粒分别注射至小鼠骨骼肌,在注射后每10 d记录小鼠累积增重,采血并用RIA法测定血清胰岛素样生长因子Ⅰ（IGF-Ⅰ）浓度,结果pCMVSP-GRF组累积增重、IGF-I浓度分别高于生理盐水组、pCMV-GRF组和pSP-GRF组（P<0.05）.结果表明：CMV与SP两种启动子组合,在小鼠骨骼肌内使外源基因表达效率提高.本研究为提高外源基因在肌肉组织的表达提供了新的途径.     

小鼠3T3-L1前脂肪细胞系的特异性标记(英文)

用增强绿色荧光蛋白特异性标记小鼠 3T3 L1前脂肪细胞系 .构建paP2 promoter EGFP载体 ,电穿孔转染小鼠 3T3 L1前脂肪细胞 ,显微荧光观察和RT PCR确认aP2基因的内源表达 .EGFP基因转入 3T3 L1前脂肪细胞 ,观察到细胞分化过程中EGFP表达和脂肪积累 .RT PCR分析表明 ,EGFP代表了稳定而真实的aP2基因的内源性表达 .建立了由脂肪组织特异表达基因aP2的表达控制的EGFP标记的小鼠 3T3 L1前脂肪细胞系 ,目前尚未见用同样方法对前脂肪细胞进行特异性标记 .该细胞系将为脂肪细胞分化机理研究以及为抗肥胖症和抗糖尿病药物筛选提供有力工具 .     

EGFP—pBABE逆转录病毒载体的构建及鉴定

目的构建表达增强绿色荧光蛋白（enhancedgreenfluorescentprotein,EGFP）的EGFP—pBABE逆转录病毒载体并对其表达进行鉴定。方法从pEGFP—N3质粒上切下EGFP片段,连接到pBABE载体,构建EGFP—pBABE重组质粒;将该重组质粒转染到Fr67细胞后,荧光显微镜下观察EGFP的表达;收集逆转录病毒感染宫颈癌SiHa细胞后荧光显微镜下观察EGFP的表达。结果成功构建EGFP—pBABE重组质粒。该质粒转染PT67细胞24h后,荧光显微镜下可观察到EGFP的表达;用该重组质粒包装的逆转录病毒感染宫颈癌SiHa细胞36h后,在荧光显微镜下可观察到明显的EGFP表达。结论成功构建表达EGFP的EGFP—pBABE逆转录病毒载体,为进一步利用该载体制备逆转录病毒并观测逆转录病毒感染细胞的效率奠定了良好的实验基础。     

甘菊DFL::EGFP高效融合蛋白表达载体的构建

增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)是一种优化的突变型GFP,DFL是从甘菊中分离出的LFY基因的同源序列。为了研究DFL基因的功能和表达模式,研究利用小片段克隆法将linker序列插入到EGFP基因5′端启始密码子前面,在pBI121载体的CaMV35S启动子的3′端后面插入一段多克隆位点,成功地构建了pBI-DFL-EGFP表达载体。通过设计特异引物,利用PCR技术扩增得了到拟南芥LFY基因的启动子序列,用粘性末端PCR技术将pBI-DFL-EGFP表达载体中CaMV35S启动子替换成LFY基因启动子,构建成了pLFY-DFL-EGFP表达载体。用含有pBI-DFL-EGFP和pLFY-DFL-EGFP质粒的农杆菌侵染洋葱表皮细胞,在荧光显微镜下分别用蓝光激发,均观测到了荧光。这一结果表明,融合蛋白DFL∷EGFP表达载体构建成功,同时还证明了通过PCR技术克隆到的LFY启动子序列具有启动子功能。     

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。     

piggyBac转座子介导的转基因叉尾斗鱼的构建

为探讨piggyBac转座子在鱼类动物中应用的可能性,以包含家蚕(Bombyx mori)肌动蛋白3启动子驱动的增强型绿色荧光蛋白(enhance green fluorescent protein,EGFP)基因的piggyBac质粒为载体,以及一个包含piggyBac转座酶的辅助质粒,采用显微注射的方法将其导入叉尾斗鱼(Macropodusopercularis)受精卵中,利用PCR技术证实了piggyBac转座子能够介导EGFP基因进入叉尾斗鱼基因组,并能够稳定遗传到下一代,符合孟德尔遗传规律。EGFP基因遗传到G1代的阳性鱼占交配鱼比率,即外源基因整合率为12.30%。实验证明,piggyBac质粒有可能成为水产动物转基因实验的新型载体。     

对虾白斑综合征病毒ORF220基因在昆虫细胞内的表达及其分布

对虾白斑综合征病毒厦门分离株ORF220编码真核生物GP130受体同源蛋白.将ORF220和绿色荧光蛋白编码基因融合在一起克隆到昆虫杆状病毒表达载体pFastBacI,然后与AcBacmid共同转染DH10B细胞.用PCR鉴定含有ORF220和EGFP基因的重组质粒,提取纯化重组质粒并转染昆虫细胞进行表达.结果发现,DNA转染后3-5d可以在荧光显微镜下观察到绿色荧光,表明融合蛋白在昆虫系统内成功表达.用病毒上清液感染昆虫细胞进行时相观察,结果表明,ORF220蛋白在昆虫细胞的细胞质和细胞核内呈随机分布,没有特异的细胞定位.     

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。     

Factors related to successful sperm microinjection of hamster eggs: The effect of sperm species, technical experience, needle dimensions, and incubation medium on egg viability and sperm decondensation following microinjection

A series of experiments was designed to identify factors in a sperm microinjection system that could influence egg viability and decondensation of sperm nuclei after microinjection. Egg viability and sperm decondensation rates were not different among eggs microinjected with rodent sperm. The microinjection of ram sperm required a larger diameter needle for injection, which resulted in low egg viability and sperm decondensation in the first 3 mo of the study but improved greatly after 9 mo of technical experience. The degree of technical experience (3 vs 9 mo) also improved (P<0.05) egg viability after microinjection with rodent sperm; however, the rate of sperm decondensation remained unaffected. Altering the dimensions of the injection needle from a tapered needle barrel to a more uniform needle barrel increased egg viability from 61 to 96% and sperm decondensation from 3 to 27%. The use of medium 199 for incubating microinjected eggs further increased (P<0.05) the percentage of eggs containing decondensed sperm nuclei (52%) compared to eggs incubated in Holmes defined medium (28%). By altering the dimensions of the injection needle, by selecting an appropriate incubation medium, and by gaining technical experience in microinjection, the efficiency of a sperm microinjection system was improved for both rodents and domestic animals.     

Development of a heat shock inducible and inheritable RNAi system in silkworm

A heat shock inducible and inheritable RNA interference (RNAi) system was developed in the silkworm (Bombyx mori). RNAi transgenic silkworms were generated by injecting silkworm eggs with a piggyBac transposon plasmid carrying RNAi sequence against target gene driven by the Drosophila heat shock protein 70 (HSP70) promoter and the helper plasmid expressing piggyBac transposase. The transgenic EGFP gene and the endogenous eclosion hormone (EH) gene were chosen respectively as the target genes. In the RNAi transgenic silkworms, heat shock at 42 degrees C significantly and specifically reduced the expression of EGFP or EH gene in silkworms according to the corresponding RNAi targeting sequence but not in silkworms with the irrelevant RNAi sequence demonstrating the efficiency and specificity of the RNAi effect. Heat shock in the pupal stage hampered pupal-adult eclosion and reduced egg fertility in EH RNAi transgenic silkworms but not in the wild type or EGFP RNAi transgenic silkworms. The establishment of this heat inducible and inheritable conditional RNA interference system in silkworms provided an approach for the first time to dissect the functions of target genes in silkworms at different stages.     

家蚕转基因方法的初步研究

为建立家蚕转基因研究中切实可行的外源基因导入方法、分别用显微注射法、精子介导法、脂质体法和压力渗透法将含有绿色荧光蛋白(gfp)基因的转座子载体和辅助质粒转入到家蚕的受精卵中。在后代中检测到发绿色荧光的蚕茧,用PCR方法检测到后代个体染色体中含有gfp基因,并比较了上述几种方法的优缺点,为进一步进行转基因家蚕的研究奠定了基础。     

Comparison of transformation efficiency of piggyBac transposon among three different silkworm Bombyx mori Strains

The transformation rate of three different strains of silkworm Bombyx mori was comparedafter the introduction of enhanced green fluorescence protein (EGFP)-encoding genes into the silkwormeggs by microinjection of a mixture of piggyBac vector and helper plasmid containing a transposase-encodingsequence.Although there were no significant differences among the three strains in the percentages offertile moths in microinjected eggs (P=0.1258),the percentages of G_0 transformed moths in fertile mothsand injected eggs were both significantly different (P=0.01368 and P=0.02398, respectively).Thetransformation rate of the Nistari strain (Indian strain) was significantly higher than that of the other twostrains,Golden-yellow-cocoon (Vietnamese strain) and Jiaqiu (Chinese strain),which had similar rate. Theseresults indicate that the transformation efficiency of the piggyBac-based system might vary with silkwormstrains with different genetic backgrounds.The presence of endogenous piggyBac-like elements might bean important factor influencing the transformation efficiency of introduced piggyBac-derived vectors,andthe diverse amount and activation in different silkworm strains might account for the significant differences.     

Expression of the hIGF-I gene driven by the Fhx/P25 promoter in the silk glands of germline silkworm and transformed BmN cells

The expression of the human insulin-like growth factor (hIGF-I) gene driven by the Fhx/P25 promoter in the silk glands of transgenic silkworms (Bombyx mori) and in transformed silkworm cells, was achieved using BmN cells transfected with a piggyBac vector, pigA3GFP-Fhx/P25-hIGF-ie-neo containing a neomycin-resistance gene (neo), a green fluorescent protein gene (gfp), an hIGF-I gene, and a helper plasmid containing the piggyBac transposase sequence under the control of the B. mori actin 3 (A3) promoter. We selected stably transformed BmN cells expressing hIGF-I using the antibiotic G418. The expression level of hIGF-I was about 450 pg in 3 × 10(6) cells, determined by ELISA. The piggyBac vector was transferred into the silkworm eggs using sperm-mediated gene transfer. The expression level of hIGF-I per gram fresh posterior silk glands of G4 transgenic silkworms was approx. 150 ng.     

家蝇微生物感染对RNA干扰GNBP3基因的作用

为了研究家蝇Musca domestica微生物感染对RNA干扰GNBP3基因的作用,评价微生物感染后对下游抗菌肽水平的影响,本研究用RNA干扰技术沉默家蝇GNBP3基因探索最佳沉默时间及效果,检测RNA干扰后家蝇幼虫存活及化蛹情况,通过微生物喂食途径感染,采用实时荧光定量PCR方法检测抗菌肽基因(cecropins、...     

Genetic transformation of the codling moth, <Emphasis Type="Italic">Cydia pomonella</Emphasis> L., with <Emphasis Type="Italic">piggyBac</Emphasis> EGFP

Genetic transformation of the codling moth, Cydia pomonella, was accomplished through embryo microinjection with a plasmid-based piggyBac vector containing the enhanced green fluorescent protein (EGFP) gene. Sequencing of the flanking regions around the inserted construct resulted in identification of insect genomic sequences, not plasmid sequences, thus providing evidence that the piggyBac EGFP cassette had integrated into the codling moth genome. EGFP-positive moths were confirmed in the 28th and earlier generations post injection through PCR and Southern blot analyses, indicating heritability of the transgene.