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排序方式: 共有42条查询结果,搜索用时 15 毫秒
1.
本研究通过把构建的短发夹结构RNA(shRNA)真核表达载体导入鸡胚,检测鸡胚性腺分化期质粒载体在鸡胚体内代谢情况及雌性鸡胚性腺中芳香化酶基因(CYP19A1)mRNA表达效率,进而探讨利用该方法在鸡胚体内进行特定基因干涉的可行性.实验针对CYP19A1基因构建了4条特异性表达载体,一条非特异性表达载体.每个实验组选取45个新鲜种蛋作为实验材料进行胚盘下腔注射,并设立空白对照组.鸡胚发育12d时,检测各处理组鸡胚肝脏组织中质粒存在情况,并取其左侧性腺组织进行目标基因的荧光定量分析.研究结果表明,导入组在12日胚龄时所有鸡胚基因组中均可检测到绿色荧光蛋白基因(EGFP);荧光定量结果显示,导入特异性表达载体cyp-580、cyp-1083和cyp-1295后,对应雌性鸡胚性腺CYP19A1 mRNA表达效率显著低于空白对照组,干涉效率分别为:73%、52%和85%;特异性表达载体cyp-1403组CYP19A1mRNA表达效率与空白对照组相比有所降低但无显著性差异.本实验为诱导鸡胚性反转提供了新方法并建立了鸡胚发育期特定基因体内干涉新平台.  相似文献   
2.
端粒酶RNA亚基的shRNA对细胞端粒酶活性影响的研究   总被引:1,自引:1,他引:0  
[目的]探讨端粒酶RNA亚基(chTR)的短发RNA(shRNAshRNA对MDCC-MSB1细胞端粒酶活性的影响。[方法]设计合成chTRshRNA并构建表达载体,转染MDCC-MSB1细胞,应用改良TRAP法检测端粒酶活性。[结果]与对照组相比,转染24 h后,各组端粒酶活性变化不明显;转染48 h后,端粒酶活性均显著地下降,且针对模板区设计的干扰载体pSi-chTR-sh1转染后抑制效果最明显。[结论]chTRshRNA表达载体能够有效地抑制MDCC-MSB1细胞的端粒酶活性。  相似文献   
3.
为验证腺病毒载体在鸡胚成纤维细胞(CEF)及鸡胚中递送小干扰RNA的可行性,本实验利用共转染技术将表达针对新城疫病毒(NDV)NP基因shRNA的重组质粒同源重组到pGSadeno腺病毒载体系统,用重组腺病毒感染CEF和鸡胚,通过红荧光报告基因的表达情况和荧光定量PCR检测NP基因mRNA的表达对其进行鉴定,并通过细胞形态观察、血凝价的测定评价其在CEF和鸡胚上对NDV增殖的影响。实验结果显示重组腺病毒能够感染CEF,感染CEF后6h、9h、12h与对照组比较NP基因mRNA的表达量分别降低了3.2倍,25.6倍,2.57倍,并能够推迟NDV致细胞病变效应,抑制NDV在鸡胚上的增殖,延缓鸡胚的死亡。本实验构建的重组腺病毒能够在CEF和鸡胚上递送特异的shRNA,为在CEF中的RNA干涉研究开辟了新的递送途径。  相似文献   
4.
AIM: To investigate the effects of Rho-associated coiled-coil protein kinase-1 (ROCK1) and ROCK2 on apoptosis induced by hypoxia in rat cardiomyocytes. METHODS: Rat cardiomyocytes were cultured primarily and identified using an antibody targeting α-actin of striated muscle. ROCK1-shRNA and ROCK2-shRNA were transiently transfected into the cells by liposome. After 48 h, these cells were subject to hypoxia for 6 h. The cells were divided into 5 groups: blank control group, hypoxia group, hypoxia+negative control shRNA group, hypoxia+ROCK1-shRNA group and hypoxia+ROCK2-shRNA group. The beating frequency and rhythm of the cardiomyocytes were assessed by microscopy. The activity of lactate dehydrogenase (LDH) in the cell culture supernatants was detected by automatic biochemical analyzer. The cell survival rate was analyzed by the method of MTT. The cell apoptotic rate was assessed by flow cytometry. Western blotting was used to determine the expression of ROCK1, ROCK2, caspase-3 and p-PI3K. RESULTS: The primary culture of the cardiomyocytes was successful. Western blotting results showed that the transfection of ROCK1-shRNA or ROCK2-shRNA decreased the expression of ROCK1 or ROCK2 in the cardiomyocytes. Hypoxia slowed down the beat frequency of the cardiomyocytes, also made the rhythm disorder. Hypoxia increased the release of LDH and decreased the cell survival rate. Flow cytometry results showed that hypoxia increased the cell apoptotic rate. Hypoxia increased the expression of caspase-3 and decreased the expression of p-PI3K. Transfection of ROCK1-shRNA and ROCK2-shRNA into the cardiomyocytes reduced all the effects of hypoxia mentioned above. CONCLUSION: Down-regulation of ROCK1 and ROCK2 expression suppresses the apoptosis of rat cardiomyocytes induced by hypoxia. The mechanism is associated with the inhibition of caspase-3 activation and the up-regulation of p-PI3K expression.  相似文献   
5.
试验旨在克隆猪SMYD3(SET and MYND domain-containing protein 3)基因并对其进行序列分析,研究其对猪成纤维细胞增殖的影响。首先克隆猪SMYD3基因,根据其他物种SMYD3基因siRNA和shRNA序列,经同源性比对分析,获得两条猪SMYD3基因shRNA序列,分别构建pSicoR-GFP-SMYD3 shRNA1/shRNA2表达载体,转染HEK293T细胞,利用实时荧光定量PCR分析干扰效率,筛选出抑制效率较好的shRNA,并构建pLVX-IRES-ZsGreen1-SMYD3及pSicoR-GFP-SMYD3 shRNA真核表达载体,同时分析SMYD3基因对猪成纤维细胞的增殖作用,检测细胞Nanog、DNMT1及DNMT3a基因表达情况。结果显示,试验克隆得到1 404 bp的猪SMYD3基因编码区序列,生物信息学分析发现,德保猪SMYD3基因与野猪、山羊和野耗牛相应氨基酸序列的同源性分别为99.5%、93.8%和92.9%。shRNA1/shRNA2均能显著抑制SMYD3基因表达(P<0.05),抑制效果分别是34%和54%,选择pSicoR-GFP-SMYD3 shRNA2进行后续研究。通过脂质体转染法将构建的pLVX-IRES-ZsGreen1-SMYD3及pSicoR-GFP-SMYD3 shRNA真核表达载体导入HEK293T细胞,均可观察到清晰的绿色荧光。慢病毒感染细胞及实时荧光定量PCR结果显示,与空白对照组及阴性对照组相比,过表达SMYD3基因促进猪成纤维细胞增殖,Nanog和DNMT1基因表达显著升高(P<0.05);抑制SMYD3基因表达,细胞增殖受到抑制,Nanog、DNMT1、DNMT3a基因表达显著降低((P<0.05),说明SMYD3基因的表达与猪成纤维细胞的增殖显著相关。  相似文献   
6.
《中国兽医学报》2019,(1):14-20
RNAi技术可以特异性剔除或关闭特定基因的表达,使机体具有抗病毒的功效,本试验针对猪繁殖与呼吸综合征病毒(PRRSV)Nsp9基因设计多对shRNA序列,并从细胞水平验证其干扰效果,从中筛选可以使Nsp9基因沉默的优势shRNA序列。利用BLOCK-iTTM RNAi Designer软件设计并合成shRNA对应的DNA序列-ds Oligo,构建入门载体pENTR/U6/shRNA;采用共转染技术,经荧光显微镜观察、流式细胞仪检测及Real-time RT-PCR分析等方法筛选优势干扰序列。结果显示:成功构建的6对pENTR/U6/Nsp9-shRNA均具有抑制Nsp9基因表达的效果,其中pENTR/U6/Nsp9-4和pENTR/U6/Nsp9-6的抑制效果更为突出,同时成功构建1对无意义对照pENTR/U6/con-shRNA。结果表明:pENTR/U6/Nsp9-4和pENTR/U6/Nsp9-6两株优势干扰序列的成功筛选可为构建具有干扰PRRSV复制效果的稳定细胞系以及靶向PRRSV的siRNA的转基因动物提供素材。  相似文献   
7.
采用幔病毒(LV)载体介导siRNA感染宿主细胞,研究其对内源性生长抑素(Somatostatin,SS)的抑制作用.首先将筛选出的psh2与辅助质粒共转染293T细胞包装获得假病毒颗粒LV-sh2,同时,包装产生LV-sh0和LV-GFP作为阴性对照.超速离心,梯度稀释法测定病毒滴度,结果提示所构建的重组病毒滴度约为...  相似文献   
8.
慢病毒介导的小鼠生长抑素shRNA序列的设计和筛选   总被引:1,自引:0,他引:1  
生长抑素(SS)是一种多功能的脑肠肽,在动物生长轴中主要作为生长激素(GH)的负性调控因子。本研究通过慢病毒表达质粒介导shRNA,瞬时转染自身表达SS的BHK-21细胞,筛选出了靶向小鼠SS的最有效的siRNA序列。首先,分别在小鼠SS基因mRNA的246、433和539bp处找到潜在靶位点,合成3条siRNA转录模板的发夹结构以及1条阴性对照,体外退火后插入pshRNA-copGFP lentivector构建重组质粒。然后,通过对转染BHK-21细胞的荧光观察、Real-time PCR、免疫组化以及RIA等检测,转染细胞SS在mRNA水平及蛋白水平均检测到不同程度的抑制(P<0.05)。其中,psh2(SS 433~451)表现出了最高的沉默效率(转染效率68.9%时,mRNA水平的沉默效率达59.3%,P<0.05;蛋白质水平的沉默效率达55.6%,P<0.05)。本研究为降低SS在动物体内的分泌,相应提高GH的浓度,进而为促进动物生长的研究奠定了基础。  相似文献   
9.
This study was designed to clone cDNA of goat DNA methyltransferase 1(DNMT1) gene,to screen an effective shRNAproducing vector targeting goat DNA methyltransferase 1 and to improve the developmental competence of goat nuclear transfer embryos by decreasing the DNMT1 expression in donor cells.In this study,PCR primers were designed against regions of high homology between bovine and sheep sequences and then used to amplify the larger portions of the coding regions.Next,3 RNAi oligonucleotides were designed based on the cloned sequences and inserted into pRNAT-U6.1/Neo vector,acquiring 3 new vectors,respectively termed pRNAD1,pRNAD2 and pRNAD3.Then the positive cells were sorted by flow cytometry after transfection and detected by real-time PCR analysis and sodium bisulfite genomic sequencing.Finally,the developmental rates of nuclear transfer(NT) embryos generated using donor cells with and without the effective shRNA vector respectively,as well as in vitro fertilization(IVF) embryos were observed and recorded.The results showed that the coding regions of goat DNA methyltransferase 1 gene was successfully cloned(GenBank no.FJ617538).Furthermore,an effective interfering shRNA(pRNAD2) was obtained,with its interference effect being 47.88%.Finally,NT embryos with shRNA vector harbored better developmental competence during morula and blastocyst stage compared to controls(P 〈 0.05),reaching the similar rates to IVF embryos(P 〉 0.05).In conclusion,goat DNA methyltransferase 1 gene cDNA was cloned and sequenced,an effective shRNA vector responsible for inhibiting DNA methyltransferase 1 expression was developed and the developmental competence of goat nuclear transfer morulae and blastcysts was significantly improved,which provided a feasible pathway for improving goat nuclear transfer embryo development competence by decreasing the methylation level in donor cells through RNAi-mediated manner.  相似文献   
10.
利用shRNA(Small hairpin RNA)载体调控基因表达已经成为研究基因功能的有力工具。针对增强型绿色荧光蛋白标记基因(Enhanced green fluorescent protein,egfp),分别设计茎部长度为21bp、27bp、29bp的干扰片段,体外合成的单链shRNA片段退火后连入带有U6启动子的真核表达载体psiSTRIKE中,酶切及测序结果证明设计的干扰片段已经成功克隆进入表达载体,为进一步在小鼠细胞以及个体水平上综合评价不同茎部长度shRNA的干扰效应奠定了基础。  相似文献   
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