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．     

Production of transgenic calves by somatic cellnuclear transfer

Bovine fetal oviduct epithelial cells were transfected with constructed double marker selective vector(pCE-EGFP-IRES-Neo-dNdB) containing the enhanced green fluorescent protein (EGFP) and neomycin-resistant(Neo^r) genes by electroporation, and a transgenic cell line was obtained. Somatic cell nuclear transfer (SCNT) was cartied out using the transgenic cells as nuclei donor. A total of 424 SCNT embryos were reconstructed and 208 (49.1%) of them developed to blastocyst stage. 17 blastocysts on D 7 after reconstruction were transferred to 17 surrogate calves,and 5 (29.4%) recipients were found to be pregnant. Three of them maintained to term and delivered three cloned calves.PCR and Southern blot analysis confirmed the integration of transgene in all of the three cloned calves. In addition, expression of EGFP was detected in biopsy isolated from the transgenic cloned calves and fibroblasts derived from the biopsy. Our results suggest that transgenic calves could be efficiently produced by SCNT using transgenic cells as nuclei donor. Furthermore, all cloned animals could be ensured to be transgenic by efficiently pre-screening transgenic cells and SCNT embryos using the constructed double marker selective vector.     

原核显微注射产生转基因绵羊胚胎

体外采集绵羊卵丘卵母细胞复合体,成熟培养24 h,经过体外受精培养17 h,比较高速离心对胚胎发育的影响;高速离心可以使黑色脂滴甩到一边从而使受精卵原核清晰可见.然后将绵羊乳腺特异表达人肝细胞再生增强因子和真核细胞表达增强绿色荧光蛋白(Enhanced Green fluorescence protein,EGFP)的载体DNA显微注射于绵羊受精卵雄原核中,并将异构胚在SOF液中发育培养.结果表明:高速离心组囊胚率低于对照组,但是没有显著性差异(P》0.05);显微注射外源基因2天后在激光共聚焦显微镜下可见荧光胚胎;PCR检测5个荧光胚胎均可见特异性条带.在原核显微注射生产转基因胚胎中,绿色荧光蛋白可作为标记基因进行早期胚胎筛选,为提高转基因动物移植效率奠定实验基础.     

热处理对桑蚕丝纤维结构与性能的影响

文章以普通桑蚕丝纤维和经壳聚糖处理的桑蚕丝纤维为研究对象,对其进行热处理,研究热作用对其结构与性能的影响.研究结果表明,两种真丝纤维经热处理后均产生失重与泛黄现象,且壳聚糖处理真丝纤维较普通桑蚕丝更明显.热处理后,普通桑蚕丝纤维结晶度提高,壳聚糖处理真丝纤维结晶度下降,同时其纤维表面出现皱缩条纹.经壳聚糖处理的真丝纤维更易老化.     

表达FLP重组酶pCEP4-FLP载体的构建及其在FLP/FRT重组系统中的应用

构建重组载体pCEP4-FLP和pEF1a-FRT-stop-FRT-GFP,重组载体pEF1a-FRT-stop-FRT-GFP瞬时转染PK-15细胞24h后,再将重组载体pCEP4-FLP瞬时转染该细胞,利用荧光显微镜观察PK-15细胞GFP蛋白的表达情况,利用细胞流式技术统计绿色荧光蛋白表达效率.结果表明:重组载体pCEP4-FLP在细胞内能够表达FLP重组酶,并且表达的FLP重组酶能够识别FRT位点,删除两同向FRT位点间的DNA片段,为FLP/FRT位点特异性重组系统在转基因猪的应用奠定了基础.     

绵羊乳腺特异表达人肝细胞再生增强因子载体的构建及表达

用PCR技术分别从人和绵羊基因组DNA中扩增人肝细胞再生增强因子(Human augmenter of liver regeneration,hALR)基因和绵羊β-乳球蛋白(sheep beta-lactoglobulin,BLG)基因启动区序列,从pEGFP-C1质粒中扩增增强绿色荧光蛋白(Enhanced Green fluorescence protein,EGFP)基因及其表达调控元件．由质粒p7zf( )构建成ALR基因乳腺特异表达、EGFP基因非组织特异性表达载体．同时体外培养绵羊胎儿成纤维细胞(sheep fetal fibroblast cells,SFFCs),脂质体介导载体DNA转染SFFCs,激光共聚焦显微镜观察和PCR检测转基因细胞,结果表明,增强绿色荧光蛋白基因在SFFCs中表达,转基因细胞中可扩增出BLG、ALR、EGFP基因条带．     

Controlled expression of enhanced green fluorescent protein and hepatitis B virus precore protein in mammalian cells

A novel tetracycline regulation expression system was used to regulate the expression of enhanced green fluorescent protein (EGFP) and hepatitis B virus precore protein in the mammalian cell lines with lipofectAMINE. Flow cytometry assays showed that application of the system resulted in about 18-fold induction of EGFP expression in CHO cell lines and 5-fold induction in SSMC-7721 cells and about 2-fold in the HEK293 cells. Furthermore, the effective use of this system for the controlled expression of HBV precore protein gene in hepatocellular carcinoma cells was tested.     

Transgene directionally integrated into C-genome of Brassica napus

Transgenic Brassica napus has been widely planted in Canada, the United States, and some other countries. In China, although the policy for genetically modified foods has not yet opened, genetically modified rape- seed oil as raw material for biodiesel of…     

Mutant acetolactate synthase （ALS） gene as a selectable marker for Agrobacterium-mediated transformation of soybean

Soybean is one of the crops most difficult to be manipulated in vitro. Although several soybean marker genes, all the selectable markers used were from bacteria origin. To find suitable selectable marker gene from plant origin for soybean transformation, a mutant acetolactate synthase （ALS） gene from Arabidopsis thaliana was tested for Agrobacterium-mediated soybean embryo axis transformation with the herbicide Arsenal as the selective agent. Transgenic soybean plants were obtained after the herbicide se- lection and the To transgenic lines showed resistance to the herbicide at a concentration of 100 g/ha. ALS enzyme assay of To transgenic line also showed higher activity compared to the wild type control plant. PCR analysis of the T1 transgenic lines confirmed the integration and segregation of the transgene. Taken together, our results showed that the mutant ALS gene is a suitable selectable marker for soybean transformation.     

质粒pAy6.8与家蚕受精卵核基质的体外结合

体外结合实验表明，携带天蚕丝素基因核心区的质粒ｐＡｙ６．８可与家蚕受精卵核基质紧密结合，而且同内源ＭＡＲ片段竞争结合位点．这为通过外源质粒而构建转基因家要提供了理论依据，并提示转移质粒在家蚕受体中的复制和传代可能与核基质有关．     

增强型绿色荧光蛋白基因真核表达载体的构建

构建增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)基因的真核表达载体pCDNA3．1( )-EGFP,转染至培养的Hela细胞中成功表达,并发出绿色荧光,证明EGFP一种良好的报告基因和筛选标记．     

家蚕卵蛋白的 SDS-PAGE 分析(动物科学)

本研究探讨了蛋白质提取方法、十二烷基磺酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)条件对家蚕卵蛋白分离效果的影响。分别用裂解液提取法、TCA沉淀法、冷丙酮沉淀法和TCA-丙酮沉淀法制备蛋白质样品,然后进行6%～12%、6%～15%两种梯度胶的SDS-PAGE,获得了不同提取方法和电泳条件下的电泳图谱用以比较家蚕卵蛋白分离效果。结果表明用TCA-丙酮沉淀法除盐,再用含9 mol.L-1尿素、4%CHAPS、1%DTT的裂解液重悬,最适合家蚕卵蛋白提取;6%～12%梯度胶对家蚕卵蛋白的分离较为适宜。利用这一优化方法对家蚕感染微孢子虫的蚕卵和正常蚕卵进行了比较研究,在分子量66 kD和85 kD处找到了与感染微孢子虫相关的蛋白,为进一步进行正常蚕卵和感染微孢子虫蚕卵的蛋白质组学研究奠定了基础。     

抗稻瘟病和纹枯病的转基因水稻新品系

将水稻碱性几丁质酶基因（RC24）导入优良灿稻品种竹籼B,外源RC24基因可以稳定整合到RO代至R6代转基因水稻基因组中,并得到表达,已获得同时抗稻瘟病和纹枯病的转基因品系竹转68和竹转70以及43个转基因纯合株系。     

Generation of selectable marker-free transgenic rice resistant to chewing insects using two co-transformation systems

To produce selectable marker-free (SMF) transgenic rice resistant to chewing insects, the Bacillus thuringiensis cryIA(c) gene (Bt) was introduced into two elite japonica rice varieties by using two Agrobacterium-mediated co-transformation systems. One system is with a single mini-twin T-DNA binary vector in one Agrobacterium strain, which consists of two separate T-DNA regions, one carrying the Bt while the other contains the selectable marker gene, hygromycin resistant gene (HPT). The other system uses two separate binary vectors in two separate Agrobacterium cultures, containing the Bt or HPT gene on individual plasmids. A lot of independent transgenic rice lines harboring both Bt and selectable marker genes were obtained. The results showed that the co-transformation frequency of the Bt gene and HPT gene was much higher by using the mini-twin T-DNA vector system (29.87%) than that by the two separate binary vector systems (4.52%). However, the frequency of the SMF transgenic rice plants obtained from the offspring of co-transgenic plants (21.74%) was lower for the mini-twin T-DNA vector system than that for the latter (50-60%). The data of ELISA implied that the expressed Bt proteins were quantitated as 0.025-0.103% of total leaf soluble proteins in the transgenic plant. Therefore, several elite transgenic rice lines, free of the selectable marker gene, were chosen. The results from both in vitro and in vivo insect bioassays indicated that the SMF transgenic rice was shown to be highly resistant to the striped stem borer and rice leaf folder. Moreover, in a natural field condition without any insecticide applied, all the transgenic rice plants were found to be not injured by the rice leaf folder, whereas the wild types were impaired seriously.     

家蚕新的非编码RNA功能初探

构建了家蚕50～500 nt非编码RNA的cDNA文库,发现了189个新的ncRNA,其中一个小RNA-Bm-86在家蚕幼虫和蛹中的表达量显著高于卵和成虫.利用生物信息学软件对该ncRNA的特性及其与上下游基因的关系进行了深入分析,并利用Northern杂交和半定量RT-PCR技术对该ncRNA与其宿主基因在家蚕不同组织的表达情况进行了研究.结果发现,该ncRNA属于H/ACA box snoRNA家族,是由家蚕eIF5A基因的第二个内含子转录产生的,且在家蚕中没有明显的靶标位点.分析其上下游基因结构发现,在其上游263 bp处存在着一个可能的启动子位点,表明其有独立转录的倾向.进一步分析Bm-86与其宿主基因eIF5A在家蚕不同组织部位的表达情况发现,二者在表达上存在着负相关的关系,且该现象在丝腺中尤其明显.推测Bm-86可能通过影响eIF5A基因的表达参与到家蚕丝腺发育调控过程中.     

转BpmiR156基因白桦株系的耐盐性分析

【目的】探究植物年龄响应因子miR156的耐盐功能。【方法】以BpmiR156过表达白桦株系和非转基因对照株系为材料,通过PCR及qPCR分析各转基因株系中外源BpmiR156的稳定性及表达情况;同时对转基因及对照株系进行NaCl胁迫处理,调查胁迫后盐害指数、生理指标及生长速度。【结果】各转基因株系中外源BpmiR156已整合到白桦基因组中,且表达量显著高于非转基因对照株系;NaCl胁迫后,转基因株系的H₂O₂、丙二醛(MDA)含量及盐害指数高于对照株系,生长速度变慢。【结论】BpmiR156基因在白桦中稳定表达,BpmiR156基因过表达白桦株系与对照株系相比,在盐胁迫后盐害指数增加,生长速度变慢,膜系统损伤更严重,说明转入miR156基因在一定程度上降低了白桦的耐盐性。     

Antigen-induced apoptotic death of Ly-1 B cells responsible for autoimmune disease in transgenic mice.

Studies on transgenic mice expressing immunoglobulins against self-antigens have shown that self-tolerance is maintained by active elimination (clonal deletion), functional inactivation (clonal anergy) of self-reactive B cells, or a combination of both. We have established and characterized a transgenic mouse line expressing an anti-erythrocyte autoantibody. In contrast to other autoantibody transgenic lines, about 50% of the animals of this transgenic line suffer from autoimmune disease, indicating a loss of self-tolerance. Here we show that peritoneal Ly-1 B cells (also known as B-1 cells) are responsible for this autoimmune disease in our transgenic mice. A few self-reactive Ly-1 B cells that have somehow escaped the deletion mechanism expand in the peritoneum because of the absence of self-antigen. These Ly-1 B cells are eliminated in vivo by apoptosis once exposed to self-antigen. On the basis of these results we propose a novel autoantibody production mechanism whereby self-reactive B cells sequestered in compartments free of self-antigens may survive, proliferate and be activated for generation of pathogenic autoantibodies in autoimmune diseases.     

Pre-B cells in kappa-transgenic mice

Recent experiments have shown that the microinjected kappa-chain gene of transgenic mice is expressed in a tissue-specific fashion only in B lymphocytes. The next step was to determine whether, within the B-lymphocyte lineage, the kappa-chain gene was expressed in a normal developmental fashion. Normally, only mu heavy(H)-chain genes, and not kappa-chain genes, are expressed in pre-B cells. To obtain cloned cell lines derived from early cells of the B-cell lineage, we transformed bone marrow cells from kappa-transgenic mice with Abelson murine leukaemia virus (A-MuLV) and tested the resultant cell lines for the retention of the kappa transgene and its expression in RNA and protein. We found that cells with the pre-B phenotype exist in kappa-transgenic mice. We further observed that in A-MuLV-transformed cell lines from a kappa-transgenic mouse with a high copy number of the transgene, the proportion of cell lines expressing kappa (transgenic kappa) was higher than in cell lines from normal or low copy number transgenic mice.     

Over-expression of an Arabi-dopsisd δ-OAT gene enhances salt and drought tolerance in transgenic rice

δ-OAT, ornithine-δ-aminotransferase, is the key enzyme involved in proline biosynthesis. In this study the Arabidopsis δ-OAT gene was transferred into rice (Oryza sativa L. ssp japonica cv. Zhongzuo 321), whose successful integration was demonstrated by PCR and Southern blot analysis. The over-expression of the gene in transgenic rice was also confirmed. Biochemical analysis showed that, under salt or drought stress conditions, proline contents in the leaves and roots in transgenic rice plants were 5- to 15-fold of those in non-transgenic controls. Under stress conditions, germinating rate of transgenic lines is higher than that of controls. Although the growth of rice plants tested were more and more retarded with the increasing of NaCI concentration, the transgenic plants grow faster compared to the controls under the same stress condition. Meanwhile, the resistance to KCl and MgSO4 stresses was also found enhanced in transgenie rice. Furthermore, the over-expression of δ-OAT also improved the yield of transgenic plants under stress conditions. The average yield per plant of transgenic lines increases about 12%--41% more than that of control line sunder 0.1 mol/L NaCI stress. These data indicated that the over-expression of δ-OAT, with the accumulation of proline, resulted in the enhancement of salt and drought tolerance and an increase of rice yield, which is of significance in agriculture.     

氯霉素对斑马鱼幼鱼发育及免疫毒性的研究

以野生型及转基因Tg(lyz:EGFP)斑马鱼幼鱼为研究对象,研究氯霉素对斑马鱼幼鱼的发育毒性及对免疫细胞的影响。用不同浓度的氯霉素分别处理72 h,观察斑马鱼幼鱼的生存率、畸形率和形态变化,以及标记绿色荧光免疫细胞在斑马鱼幼鱼中的数量。结果显示,随着氯霉素浓度(≥100 mg/L)的升高和暴露时间的延长,斑马鱼的存活率和孵化率降低,死亡率升高;氯霉素对斑马鱼幼鱼的致畸作用,主要表现为心包水肿、卵黄囊水肿、脊柱弯曲;通过对Tg(lyz:EGFP)品系的观察,发现氯霉素(≥75 mg/L)可以降低斑马鱼幼鱼免疫细胞的数量。研究表明,氯霉素对斑马鱼幼鱼的致畸和致死作用具有剂量、时间依赖性,并可以导致斑马鱼幼鱼的免疫细胞数量减少,具有免疫毒性。