毛囊特异表达载体pCDsR-UI的构建以及绒山羊胎儿成纤维细胞的体外转染和筛选

构建IGF1基因的毛囊特异表达载体,可为今后通过体细胞核移植技术建立转基因克隆绒山羊准备核供体细胞.将绵羊IGF1 cDNA序列,连接到含有UHS启动子序列的克隆载体p19TU的SalI、SphI位点,获得过渡质粒载体p19TUI;pCDsRed2和过渡质粒载体分别经酶切和连接构成表达载体pCDsR-UI.以组织块贴附法分离和培养绒山羊胎儿成纤维细胞,外源性表达载体以脂质体方法转染所培养的第2代成纤维细胞,DMEM/F12+ 10％ FBS、37℃5％CO2中培养,经添加G418筛选,获得了稳定表达红色荧光蛋白的细胞克隆,经特异性PCR鉴定证实外源基因已经整合在细胞基因组中.转基因前后分析细胞生长曲线和染色体核型证明转基因细胞的生长状态良好、各项参数正常.     

人MTERF3基因启动子的克隆及其荧光素酶报告基因载体的构建

人MTERF3基因编码线粒体基因转录和能量代谢的负调控因子。采用PCR技术对人MTERF3基因5'侧翼上游1251 bp启动子序列进行扩增,并将其克隆至荧光素酶表达载体p GL6-TA,构建人MTERF3基因启动子荧光素酶报告基因质粒。经酶切、测序鉴定后,将其用脂质体转染体外培养的HEK293细胞株,利用双荧光素酶测定系统检测其表达活性。研究结果表明,克隆获得的1251 bp DNA序列与Gen Bank报道的一致,且插入方向正确。含人MTERF3基因启动子的报告基因荧光素酶的表达活性显著提高(P0.05),约为对照组(空载体p GL6-TA)的9.8倍。本研究通过对人MTERF3基因启动子的克隆及其荧光素酶表达载体构建与表达活性的测定,为进一步阐明人MTERF3基因表达的调控机制奠定实验基础。     

精细突变小鼠模型的研究进展

基因打靶研究最重要的应用之一是研制人类疾病动物模型。人类的疾病许多是由于基因功能丧失引起的 ,也有许多是由于基因超表达或功能获取 (ｇａｉｎｏｆｆｕｎｃｔｉｏｎ)引起的。后者的情况就无法用基因剔除的方法获得相应的疾病模型。因此研究者发明了各种可以将诸如插入终止蜜码子或替换某个氨基酸之类的精细突变引入小鼠基因组中的方法。早期的研究者采用的方法主要有两种 ,一是将不含选择标记基因的打靶载体直接经显微注射法引入ＥＳ细胞 ,然后用ＰＣＲ分析鉴定同源重组克隆[1] ;另一种是用带有精细突变但无选择标记基因的打靶载体与仅…     

人MiTERF3基因启动子的克隆及其荧光素酶报告基因载体的构建

人MiTERF3基因编码线粒体基因转录和能量代谢的负调控因子。采用PCR技术对人MiTERF3基因5''侧翼上游1251 bp启动子序列进行扩增,并将其克隆至荧光素酶表达载体pGL6-TA,构建人MiTERF3基因启动子荧光素酶报告基因质粒。经酶切、测序鉴定后,将其用脂质体转染体外培养的HEK293细胞株,利用双荧光素酶测定系统检测其表达活性。研究结果表明,克隆获得的1251 bp DNA序列与GenBank报道的一致,且插入方向正确。含人MiTERF3基因启动子的报告基因荧光素酶的表达活性显著提高（P<0.05）,约为对照组（空载体pGL6-TA）的9.8倍。本研究通过对人MiTERF3基因启动子的克隆及其荧光素酶表达载体构建与表达活性的测定,为进一步阐明人MiTERF3基因表达的调控机制奠定实验基础。     

Pax5启动子的克隆及其在人淋巴瘤细胞系HL-60中转录因子的确定

克隆Pax5基因的启动子,插入荧光素酶报告基因载体中,并检测其活性;采用PCR技术从人淋巴瘤细胞系HL-60基因组中扩增出Pax5启动子,插入荧光素酶报告基因载体pGL3-basic中,确定所扩增的DNA序列,在293T细胞中检测其活性;测序结果表明扩增的Pax5启动子序列正确,活性实验表明构建的报告基因具有启动子活性,转录因子SP1和Runx1能以剂量依赖的方式提高Pax5报告基因的转录活性;克隆了Pax5启动子,并发现转录因子SP1和Runx1能够调控Pax5的转录。     

植物无标记转化的诱导表达Cre/loxP重组系统的构建

应用Cre/loxP系统位点专一性重组的特点构建诱导表达的定位重组系统,用以特异性的敲除转基因植物的标记基因.为了获得诱导表达启动子,从大豆基因组DNA中用pfu酶克隆热激蛋白启动子gmhsp17.5c,将其克隆到pUC118-HincⅡ载体并测序.结果表明,508nt的gmhsp17.5c与已报道序列(GenBank,AF544399)比较,核苷酸的同源性为99.8%.利用该诱导启动子分别构建了含gmhs p17.5c-cre基因组件和gmhsp17.5c-gus基因组的诱导型植物表达载体pC23HC和pC23HG.此外1个含有loxP-gus-loxP组件的组成型植物表达载体pC23LG被构建.通过对3个植物表达载体做多重酶切及亚克隆后测序分析表明载体pC23HC全长10947bp,载体pC23HG全长11396bp,载体pC23LG全长11900bp,符合预期设计.一套由pC23HC和pC23LG组成的植物无标记转化的诱导表达Cre/loxP重组系统被构建,为进一步将诱导表达的标记基因删除系统用于植物的无标记转化奠定基础.     

水稻petH基因启动子的克隆及其转化载体的构建

以水稻幼嫩叶片为材料,提取基因组DNA;根据GenBank公布的该启动子序列设计引物,克隆了水稻的petH启动子,并构建了用于植物转化的双元载体p1391Z-OsR-petH-pro,为进一步研究该启动子在植物体中表达调控模式提供了试验条件。     

甘草水通道蛋白PIP1基因正义和反义表达载体的构建

根据已报道的GuPIP1基因序列设计特异引物,克隆甘草水通道蛋白GuPIP1基因cDNA序列,将该片段正向、反向分别插入植物表达载体pMBW330的CaMV35S启动子和OCS终止子之间,构建了正义、反义表达载体．通过双酶切、PER和DNA测序鉴定后,分别导入农杆菌EHA105中．     

人NLK上游启动子区的克隆与鉴定

初步鉴定并分析NLK上游启动子,为研究其转录调控打下基础。对NLK基因翻译起始位点上游约1 958 bp的序列分别进行生物信息学分析,以PCR技术扩增以上序列并测序。将PCR所得到的NLK上游片段进一步克隆到PGL-3basic载体中,构建荧光素酶报告基因质粒PGL-3basic-luc。通过荧光素酶报告基因实验检测上述启动子的活性。成功构建了包含NLK基因上游启动子序列的荧光报告系统,经荧光素酶报告基因实验证明该重组质粒体具有转录活性。构建的NLK基因上游启动子报告基因载体为进一步研究NLK基因的转录调控机制奠定了基础。     

含PGK启动子慢病毒表达质粒的构建与鉴定

为了构建一个含有人源PGK1(human phosphoglycerate kinase 1)启动子的慢病毒表达载体 pL-PGK-GFP.采用PCR从人组织中扩增PGK1基因的启动子部分,再用酶切-连接的方法将扩增的启动子区片段亚克隆入慢病毒表达质粒pL-EGFP中,再用测序、酶切和瞬时表达的方法进行鉴定.结果是下游的eGFP基因在PGK1启动子驱动下,在293FT细胞中表达绿色荧光蛋白报告基因,这表明成功构建了慢病毒表达质粒pL-PGK-GFP.扩增的537bp PGK1启动子片段具有一定的启动效率,能在HIV来源的慢病毒载体中驱动下游目的基因的表达.     

miR-18a对于成肌细胞胶原蛋白表达的影响

细胞外基质是骨骼肌微环境的重要组成,基质中胶原蛋白的过量表达,会导致骨骼肌纤维化,因此研究miR-18a对于成肌细胞胶原蛋白表达的影响具有重要意义。本研究利用实时荧光定量PCR、细胞划痕、组织切片HE染色等方法检测了肌肉损伤修复模型中miR-18a及胶原蛋白基因的表达变化以及在成肌细胞C2C12中过表达miR-18a模拟物后对胶原蛋白基因表达、细胞迁移和成肌细胞转分化的影响。结果表明,miR-18a及胶原蛋白的表达量会响应骨骼肌的损伤修复过程。在成肌细胞中,miR-18a的过表达会抑制胶原蛋白相关基因的表达。但miR-18a的过表达并不影响成肌细胞的迁移和向骨的转分化。因此miR-18a对于细胞外基质相关基因的研究,可以为骨骼肌损伤修复的治疗提供理论依据。     

奶牛胎儿细胞多位点基因打靶的研究

利用奶牛rDNA基因间的ITS重复序列作为靶位点,对奶牛胎儿成纤维细胞进行多位点基因打靶,建立以重复序列为靶位点的多位点基因打靶技术,并为克隆定点转基因奶牛提供核供体。首先分离培养出奶牛胎儿成纤维细胞,并进行性别鉴定和核型分析。采用MTT比色法确定了G418和GCV正负筛选的最低有效浓度。然后通过多位点基因打靶载体转染、正负筛选获得7个表达绿色荧光的克隆细胞系,经PCR,RT-PCR和测序证实其中1个细胞系为定点整合的克隆细胞系,且GFP基因表达。     

靶向肿瘤多药耐药基因mdr1的siRNA的构建与鉴定

mdr 1基因及其表达产物P-gp是引起肿瘤细胞多药耐药(MDR)的主要原因,抑制mdr 1基因的表达可用于逆转MDR.RNAi可用于特异抑制靶基因的表达,本研究的目的是构建获得可特异有效靶向mdr 1基因的siRNA元件.应用siRNA设计软件与mRNA结构分析软件设计构建了3个分别靶向mdr 1基因mRNA环结构和茎结构的siRNA元件,同时构建了携带mdr1基因序列的luc报告质粒,通过siRNA表达质粒与携带靶序列的报告质粒的共转染抑制实验检测不同siRNA的抑制效率,结果显示靶向环结构siMDR1B具有较好的抑制效率和特异性.进一步将siMDR1B表达载体与mdr1基因表达载体共转染细胞,应用免疫流式细胞术检测显示,相比对照细胞,siMDR1B可显著抑制其转染后mdr1基因产物P-gp蛋白的表达活性.同时采用CCK-8细胞活性检测试剂评价了siMDR1B对细胞活性的影响,结果显示siMDR1B不会影响细胞活性,具有良好的特异性.本研究获得的可有效靶向mdr 1基因的siRNA元件可为进一步开展逆转MDR研究提供重要基础.     

Production of gene-targeted sheep by nuclear transfer from cultured somatic cells

It is over a decade since the first demonstration that mouse embryonic stem cells could be used to transfer a predetermined genetic modification to a whole animal. The extension of this technique to other mammalian species, particularly livestock, might bring numerous biomedical benefits, for example, ablation of xenoreactive transplantation antigens, inactivation of genes responsible for neuropathogenic disease and precise placement of transgenes designed to produce proteins for human therapy. Gene targeting has not yet been achieved in mammals other than mice, however, because functional embryonic stem cells have not been derived. Nuclear transfer from cultured somatic cells provides an alternative means of cell-mediated transgenesis. Here we describe efficient and reproducible gene targeting in fetal fibroblasts to place a therapeutic transgene at the ovine alpha1(I) procollagen (COL1A1) locus and the production of live sheep by nuclear transfer.     

沉默SEMA3A基因对胶质瘤细胞U251增殖和转移的影响

探讨了SEMA3A基因在恶性胶质瘤细胞U251迁移和侵袭能力中的作用, 及其用于胶质瘤临床治疗的可行性. 用特异识别SEMA3A基因的短发卡RNA(short hairpin RNA,shRNA)片段与真核表达载体pFH-GFP-L连接, 再与辅助质粒联用来包装慢病毒. 通过荧光显微镜观察感染胶质瘤细胞U251的感染效率. 通过实时定量PCR技术和Western-blot技术验证了U251细胞中SEMA3A基因的沉默效果. 通过MTT法、PI染色法和Transwell小室分别检测了U251细胞增殖、细胞周期和运动能力的变化. 结果表明, SEMA3A-shRNA慢病毒感染U251能有效下调SEMA3A基因的表达水平(P <0.01), 使U251细胞的增殖能力、迁移和侵袭能力明显降低(P <0.05), 细胞大量阻滞在G2/M期, 并促进了细胞凋亡(P <0.05).     

A novel peptide,selected from phage display library of random peptides,can efficiently target into human breast cancer cell

To develop a targeting vector for breast cancer biotherapy, MDA-MB-231 cell, a human breast cancer cell line, was co-cultured with pC89 （9 aa） phage display library of random peptides. In multiple inde-pendent peptide-presenting phage screening trials, subtilisin was used as a protease to inactivate extra-cellular phages. The internalized phages were collected by cell lysising and amplified in E. coli XLI-Blue. Through five rounds of selection, the pepUde-presenting phages which could be internalized in MDA-MB-231 cells were isolated. A comparison was made between internalization capacities of peptide-presenting phages isolated from MDA-MB-231 cells and RGD-integrin binding phage by coculturing them with other human tumor cell lines and normal cells. The nucleoUde sequences of isolated peptide-presenting phages were then determined by DNA sequencing. To uncover whether phage coat protein or amino acid order was required for the character of the pepUde to MDA-MB-231 cells, three peptides were synthesized. They are CASPSGALRSC, ASPSGALRS and CGVIFDHSVPC （the shifted sequence of CASPSGALRSC）, and after coculturing them with different cell lines, their targeting capacities to MDA-MB-231 cells were detected. These data suggested that the internalization process was highly selective, and capable of capturing a specific peptide from parent peptide variants. Moreover, the targeting internalization event of pepUdes was an amino acid sequence dependent manner. The results demonstrated the feasibility of using phage display library of random peptides to develop new targeting system for intracellular delivery of macromolecules, and the peptide we obtained might be modified as a targeting vector for breast cancer gene therapy.     

Folic Acid-Polyethyleneimine-Ethosome as a Potential Tumor Targeting Carrier: Preparation,Characterization and in vitro Evaluation

A novel composite carrier of folic acid( FA)-polyethyleneimine( PEI)-ethosome( Eth)( FA-PEI-Eth)was developed for the treatment of cancers through loading and targeting delivery of multidrug( including gene and other drugs) into cancer cells. Physical and chemical property tests were done to prove the grafting of the composite. Gel retardation test was done to determine the optimal ratio of DNA@ PEI complex,and cytocompatibility tests and tumor cell uptake tests were done to evaluate the efficiency of the composite. The results demonstrated that the FA-PEI-Eth could effectively deliver a gene and other drugs into tumor cells simultaneously,and suggested that this composite would be a promising carrier in tumortargeted therapy applications.     

C. elegans cell-signalling gene sem-5 encodes a protein with SH2 and SH3 domains.

The induction of the hermaphrodite vulva and the migration of the sex myoblasts in the nematode Caenorhabditis elegans are both controlled by intercellular signalling. The gonadal anchor cell induces formation of the vulva from nearby hypodermal cells, and a set of somatic gonadal cells attract the migrating sex myoblasts to their final positions. Many genes required for vulval induction have been identified, including the let-23 receptor tyrosine kinase gene and the let-60 ras gene. We report here the identification and characterization of a new gene, sem-5 (sem, sex muscle abnormal), that acts both in vulval induction and in sex myoblast migration. On the basis of its DNA sequence, sem-5 encodes a novel 228-amino-acid protein which consists almost entirely of one SH2 (SH, src homology region) and two SH3 domains. SH2 and SH3 domains are present in many signalling proteins regulated by receptor and non-receptor tyrosine kinases. Mutations that impair sem-5 activity alter residues that are highly conserved among different SH2 and SH3 domains. Our results indicate that the sem-5 gene encodes a novel protein that functions in at least two distinct cell-signalling processes.     

壳寡糖纳米载体细胞内递送EGFR脱氧核酶的研究

设计靶向EGFR mRNA的脱氧核酶(EGFR DRz),以壳寡糖(COS)为材料,建立了一种有效的纳米基因细胞内传递体系,并研究其介导的靶向EGFR的脱氧核酶在Hela细胞内的生物学效应.流式结果表明COS-EGFR DRz复合体转染效率为88.7%,与脂质体转染试剂的89.7%相比无显著差异.半定量RT-PCR结果显示,经壳寡糖纳米载体递送的EGFR DRz能有效地靶向切割Hela细胞内的EGFR mRNA,使其表达下降.进一步的流式分析显示细胞被阻滞在G0～G1期,并且出现凋亡现象,其中COS组的凋亡率为19.3%,大于对照组脂质体的凋亡率13.0%.研究表明,COS较脂质体有相似的转染效率和更低的毒性,是一种潜在的、有效的脱氧核酶递送载体.