Plcd1转基因小鼠的制备

通过制备Plcd1转基因小鼠研究Plcd1基因功能.首先逆转录PCR获得约2.2kb的Plcd1基因全长,T载体克隆后测序验证;以pMD19-T-Plcd1为模板,通过设计引物及PCR扩增引入酶切位点,与pEF6/V5-His同时进行酶切、连接,构建pEF6/V5-His-Plcd1表达载体;经真核表达验证后,酶切获得目标片段,显微注射681枚受精卵,在82只仔鼠中获得转基因阳性首建鼠15只,其中13只稳定遗传并建系.PLCD1-HIS融合蛋白在睾丸组织中表达,在皮肤组织中无表达.Plcd1转基因小鼠为Plcd1功能研究奠定了基础.     

苜蓿花叶病毒RNA_23′端cDNA转基因烟草及其抗病性研究

将本室克隆的编码复制酶基因3′端约1/2序列及其3′端非编码区的苜蓿花叶病毒中国分离株(AlMV-Ch)RNA23′端cDNA,重组到植物表达载体pROKII中,通过致瘤农杆菌(A-grobacteriumtumefaciens)介导,以叶圆片为转化材料,转化普通烟草,并获得了转基因植株.经卡那霉素抗性选择、PCR检测目的基因证明AlMVRNA23′端基因已整合到转基因烟草的基因组DNA中.转基因植物的攻毒试验表明转基因植株对苜蓿花叶病毒产生高水平的抗性.     

以精子载体法制备转基因小鼠的初步研究

将外源融合基因BaLA-HI与DOSPER脂质体等比较混合,加入获能精子悬液中,37度,5％CO2共培养0．5h,以这种处理精子作为转基因载体乾鼠的体外受精及胚胎移植,在获得的40只移植后代后,经PCR特异片段扩增和Southern杂交,共检测出2只呈相性的转基因小鼠,证明人胰 基因已在小鼠染色体上实现了整合,基因整合率为5％。     

苜蓿花叶病毒RNA2 3''''端cDNA转基因烟草及其抗病性研究

将本室克隆的编码复制酶基因3′端约1／2序列及其3′端非编码区的苜蓿花叶病毒中国分离株(AIMV-Ch)RNA：3′端cDNA，重组到植物表达载体pROKⅡ中，通过致瘤农杆菌(Agrobacterium tumefaciens)介导，以叶圆片为转化材料，转化普通烟草，并获得了转基因植株．经卡那霉素抗性选择、PCR检测目的基因证明．AIMV RNA：3′端基因已整合到转基因烟草的基因组DNA中．转基因植物的攻毒试验表明转基因植株对苜蓿花叶病毒产生高水平的抗性．     

用慢病毒载体介导产生绿色荧光蛋白（GFP）转基因小鼠

以慢病毒（lentivirus）载体为骨架，携带绿色荧光蛋白（GFP）基因的假病毒，通过小鼠受精卵卵周隙注射，将其感染小鼠受精卵，经移植于假孕母鼠后获得转基因小鼠．应用PCR、荧光显微镜观察和流式细胞仪分析等技术，证明了GFP基因的整合率达到40%以上；实时定量PCR分析结果表明转基因小鼠中GFP基因整合的拷贝数约为40；染色体荧光原位杂交分析结果显示GFP基因在小鼠染色体上的整合是随机的，并通过交配可将外源基因遗传至子代，获得的多整合位点和不同表达水平的转基因小鼠具有明显的实际应用和研究价值．文中报道的慢病毒载体介导的转基因技术为高效制备和选育高表达转基因小鼠品系提供了一种有效的途径．     

脑组织特异性表达胰岛素样生长因子-1转基因小鼠制备

目的制备脑组织特异性表达胰岛素样生长因子-1(Insulin-Like Growth Factors 1,IGF-1)转基因小鼠。方法采用精子为载体法进行基因转导,出生后小鼠PCR检测,建立转基因小鼠系,用Western blot和免疫荧光法分别检测转基因小鼠海马齿状回亚粒状区(subgranular zone,SGZ)IGF-1表达量和报告基因EGFP阳性细胞数。结果获得3只阳性小鼠,2只外源基因能稳定遗传,并建立了转基因小鼠系,转基因鼠SGZ区域IGF-1表达量显著高于正常鼠,EGFP也在此区域表达。结论成功制备IGF-1转基因小鼠。     

酒色着色菌Chromatium vinosum膜结合态氢酶基因hupSL的克隆及分析

光合细菌酒色着色菌(Chromatiumvinosum)含有Hup膜结合态氢酶.根据同科的桃红荚硫菌(Thiocapsaroseopersicina)Hup氢酶同源序列,在结构基因小亚基hups和hupC的保守框上设计1对简并引物:Ps:5’CCGACCAC(C/G)TACAACGCCTG3’和Pc:5’CG(G/C)GACATGATGTC(T/C)TCGCG3’.通过PCR扩增获得2.6kb的扩增片段,克隆后进行序列分析.结果表明,该片段包含部分的小亚基hupS序列、大亚基hupL的全序列及hupC部分序列.从已知的hupS序列选取适当位点合成引物AS2:5’CTACGACCATGTCACCGACA3’,并利用接头寡核苷酸序列P6:5’CCTTGTGAAATTGTTATCCGCT3’,通过PCR扩增获得1.2kb的扩增片段,克隆后进行序列分析.结果表明,该片段包含完整的膜结合态氢酶的小亚基基因hupS.     

pEGFP-N3-t-PA真核表达质粒的构建及其在成纤维细胞中的表达

目的利用基因工程技术克隆人组织纤溶酶原激活物（t—PA）基因并构建一种能高效、安全表达t—PA的pEGFP—N3-t—PA真核表达重组质粒,为进一步研制转基因动物奠定基础．方法采用高效Trizol试剂快速从黑色素瘤细胞中提取总RNA,RT—PCR获得t—PAcDNA,并将真核表达质粒pEGFP—N3和t—PA基因片段分别双酶切,将回收的pEGFP—N3大片段（4．7kb）与t—PA基因片段（1．1kb）重组．对pEGFP—N3-t—PA质粒进行酶切鉴定和基因测序鉴定．脂质体介导pEGFP—N3-t—PA转染成纤维细胞（NIn313）,并用RT—PCR法从mRNA水平检测t—PA的表达情况,用倒置荧光显微镜检测、分析其在NH 3T3细胞中的表达．结果成功地从黑色素瘤细胞中克隆了t—PA基因,并构建了以pEGFP—N3为载体的真核表达质粒载体,并能在真核细胞中表达、分泌t—PA．结论含t—PA基因真核表达质粒构建成功．     

Vill转基因小鼠的制备

摘要:目的　通过制备Vill转基因小鼠研究该基因的功能。 方法与结果　首先由RT-PRC方法获得全长约2605bp的Vill基因;经T载体克隆测序验证后,以克隆载体pMD19-T-Vill为模板,设计引物并引入酶切位点,将PRC扩增产物与pEF6/V5-His-LacZ同时进行酶切、连接,构建表达载体pEF6/V5His-Vill;经真核表达验证后,酶切获得含Vill基因的显微注射DNA构件;显微注射390枚受精卵后,在出生存活的77只仔鼠中获得转基因阳性GO代小鼠19只,其中16只能够稳定遗传并建系,转基因阳性小鼠外观未有明显改变。 结论　Vill转基因小鼠为该基因的功能研究准备了实验材料。     

High expression of human serum albumin in milk of trans- genie mice directed by the goat p-casein gene promoter region

We have constructed a mammary gland expression vector that contained the goat β-casein gene pro-moter, 5'upstream regulatory region, exons 1, 2, intron 1 as well as the human serum albumin (hALB) mini-gene (including the full-long sequences of hALB cDNA and its intron 1). Injection of the vector into mouse tail veins showed that the recombinant construct was expressed only in mammary glands. The vector was microinjected into the mouse fertilized eggs, followed by transferring the eggs into the foster mice. 33 F0 mice were obtained. Of the 33, 8 mice (5 , 3 ) were transgenic with hALB gene integration identified by PCR as well as Southern blot hybridization. The integration rate was 24.2% (8/33). Western blot analysis showed that 3 female transgenic mice had hALB expression in their milk. The hALB contents in milk reached 3.54, 0.21 and 3.03 g/L, respectively.     

High expression of human serum albumin in milk of transgenic mice directed by the goat β-casein gene promoter region

We have constructed a mammary gland expression vector that contained the goat β-casein gene promoter, 5′upstream regulatory region, exons 1,2, intron 1 as well as the human serum albumin (hALB) mini-gene (including the full-long sequences of hALB cDNA and its intron 1). Injection of the vector into mouse tail veins showed that the recombinant construct was expressed only in mammary glands. The vector was microinjected into the mouse fertilized eggs, followed by transferring the eggs into the foster mice. 33 F₀ mice were obtained. Of the 33, 8 mice (5♀, 3 ♂) were transgenic with hALB gene integration identified by PCR as well as Southern blot hybridization. The integration rate was 24.2% (8/33). Western blot analysis showed that 3 female transgenic mice had hALB expression in their milk. The hALB contents in milk reached 3.54, 0.21 and 3.03 g/L, respectively.     

通过遗传转化获得含多基因的水稻转基因纯合植株

利用基因枪法将含有4个不同基因的3个质粒共转化由粳稻品种鄂宜105号和鄂晚5号种子胚诱导的愈伤组织（5－10d龄）。从轰击的986块愈合组织中共再生出169株独立的转基因水稻植株（转化率为17％）。PCR／Southern blot分析显示70％以上的转基因植株含有所有4个基因。GUS组织化学分析、Western blot和或RT－PCR分析表明所有4个基因的共表达率为70％。未观察到任何质粒在整合中存在优势,转基因拷贝数也与基因表达量无关。遗传分析证实外源基因在后代植株中大多以孟德尔方式遗传。从其R1代为3：1孟德尔方式遗传的后代R2代植株中,鉴定含有3个或4个不同基因的转基因纯合植株系。PCR／Southern blot分析证实了这些转基因纯合植株系。这些系的植株具有相似的外源基因表达量。我们证实通过基因枪介导的共转化,结合常规育种方法筛选可以获得含多基因的转基因水稻纯合植株。这项技术为利用基因同时改良作用多个性状提供了一种途径。     

转人Cu,Zn-SOD基因马铃薯的研究

采用RT—PCR技术从人肝总RNA中分离扩增了490bp的人铜锌超氧化物歧化酶(hCM，Zn—SOD)基因的cDNA序列，进行了序列测定，结果和文献报道的一致；构建了CaMv35S启动子驱动hCu，Zn—SOD基因的植物表达载体PBICuSOD；用三亲交配法将重组质粒PBICuSOD转化到农杆菌LBA4404，转化马铃薯得到转基因植株。PCR和Southern—blot分析证明，hCu，Zn—SOD基因巳整合到马铃薯基因组中。Western blot分析表明，hCu，Zn—SOD基因在转基因马铃薯植株中得到表达。用NBT光还原法测定转基因马铃薯植株中的SOD酶活力，叶的总酶活为1066．6U／g(湿重，下同)，块茎的总酶活为10llU／g。     

Expression of recombinant human lysozyme in the milk of transgenic mice

Human lysozyme is a 130-aa (amino acid) alkaline polypeptide, and has both anti-bacterial and anti-viral properties which make it an important component of human natural immunity system. As a first step toward the ultimate goal of improving the anti-bacterial properties of bovine and ovine milk, a transgenic mouse that contains the genomic DNA sequence of the human lysozme gene has been generated for the first time. From 83 mice generated by microinjection, a total of 6 positive transgenic mice were identified by PCR and Southern blot. F1 mice positive for transgene in lines were also detected by PCR. This shows that transgene could be transmitted from founder transgenic mice to their offspring. Recombinant human lysozyme (rHlys) was found in the whey of 3 female positive transgenic mice by Western blot. The highest concentration of rHlys for transgenic mice was 0.2mg/mL. The antibacterial activity of the whey for transgenic mice was highly enhanced up to 0.4 times as much as that of human, while that of non-transgenic mouse was very low. Although the lysozyme activity of transgenic mice is still lower than that of human, the rHlys exhibits the same specific activity as that of human lysozyme. It provides a strong basis for further studies into the possible application of rHlys express in mammary gland.     

EGFP和ALR融合基因表达载体构建及其在HeLa细胞中的表达

从人血液中提取总DNA,利用PCR技术扩增人肝细胞再生增生因子基因,将其插入表达载体pEGFP—C1的多克隆位点中,构建增强绿色荧光蛋白（enhanced green fluorescence protein gene,EGFP）和人肝细胞再生增强因子（human augmenter of liver regeneration gene,ALR）融合基因表达载体pEGFP／ALR,并将其转染Hela细胞系,用含G418的DMEM／F12培养液筛选转基因细胞,然后利用PCR和聚丙烯酰胺凝胶电泳检测转基因细胞中ALR基因的存在及其表达,用荧光显微镜检测EGFP基因的表达．结果显示：得到了构建正确的EGFP和ALR融合基因表达载体;在转基因细胞中,PCR扩增得到1．7Kb的ALR条带,蛋白电泳得到57KD大小条带．与ALR和EGFP融合蛋白大小相符;荧光显微镜下观察到发绿色荧光的Hela细胞．在转基因细胞中,EGFP和ALR同时存在并表达,绿色荧光蛋白可作为报告基因指示目的基因的表达,从而简化了目的基因繁琐的检测手段．     

丙型肝炎病毒E2基因在转基因番茄植株中的表达

以T-E1E2为模板扩增得到丙型肝炎病毒包膜蛋白基因E2,构建该基因的植物表达载体p35s-E2．通过农杆菌介导的叶盘法转化番茄子叶．转基因番茄植株叶片总DNA的PCR、Southern blot检测结果表明,E2基因已整合进了转基因番茄植株基因组中;RT-PCR,Western blot分析证实E2基因在转基因番茄植株叶片中表达．     

Generation of transgenic wheat resistant to wheat yellow mosaic virus and identification of gene silence induced by virus infection

The plasmid containing the promoter Act1, the coat protein (cp) gene of wheat yellow mosaic virus (WYMV) and the selectable bar gene, was delivered via particle bombardment, directly into immature embryos of a wheat cultivars. PCR and PCR-RFLP were employed to screen the existence of the cp gene in T0 and T1 generations. Seeds from the positive T1 plants were sowed in fields heavily contaminated with WYMV to detect their resistance. In field trial of virus infection, one of the transgenic wheat lines, P8-T2, exhibited highly disease-resistance. Western blot and RT-PCR analysis showed that the expression level of cp gene in the resistant transgenic line was reduced greatly compared to those susceptible to WYMV infection. This provided evidence to presume that the resistance obtained by the transgenic wheat line was stimulated by the mechanism of the virus induced gene silencing.     

Ubi1 intron-mediated enhancement of the expression of Bt cry1Ah gene in transgenic maize (Zea mays L.)

The cry1Ah gene was one of novel insecticidal genes cloned from Bacillus thuringiensis isolate BT8. Two plant expression vectors containing cry1Ah gene were constructed. The first intron of maize ubiqutin1 gene was inserted between the maize Ubiquitin promoter and cry1Ah gene in one of the plant expressing vectors (pUUOAH). The two vectors were introduced into maize immature embryonic calli by microprojectile bombardment, and the reproductively plants were acquired. PCR and Southern blot analysis showed that foreign genes had been integrated into maize genome and inherited to the next generation stably. The ELISA assay to T1 and T2 generation plants showed that the expression of Cry1Ah protein in the construct containing the ubi1 intron (pUUOAH) was 20% higher than that of the intronless construct (pUOAH). Bioassay results showed that the transgenic maize harboring cry1Ah gene had high resistance to the Asian corn borers and the insecticidal activity of the transgenic maize containing the ubi1 intron was higher than that of the intronless construct. These results indicated that the maize ubi1 intron can enhance the expression of the Bt cry1Ah gene in transgenic maize efficiently