全文获取类型
收费全文 | 794篇 |
免费 | 57篇 |
国内免费 | 87篇 |
专业分类
938篇 |
出版年
2024年 | 1篇 |
2023年 | 3篇 |
2022年 | 6篇 |
2021年 | 16篇 |
2020年 | 5篇 |
2019年 | 21篇 |
2018年 | 16篇 |
2017年 | 9篇 |
2016年 | 15篇 |
2015年 | 23篇 |
2014年 | 40篇 |
2013年 | 49篇 |
2012年 | 28篇 |
2011年 | 49篇 |
2010年 | 38篇 |
2009年 | 36篇 |
2008年 | 45篇 |
2007年 | 52篇 |
2006年 | 53篇 |
2005年 | 54篇 |
2004年 | 43篇 |
2003年 | 43篇 |
2002年 | 40篇 |
2001年 | 42篇 |
2000年 | 33篇 |
1999年 | 22篇 |
1998年 | 20篇 |
1997年 | 26篇 |
1996年 | 16篇 |
1995年 | 23篇 |
1994年 | 17篇 |
1993年 | 5篇 |
1992年 | 11篇 |
1991年 | 4篇 |
1990年 | 1篇 |
1989年 | 5篇 |
1988年 | 6篇 |
1987年 | 4篇 |
1986年 | 5篇 |
1985年 | 1篇 |
1984年 | 3篇 |
1983年 | 3篇 |
1982年 | 2篇 |
1981年 | 1篇 |
1980年 | 2篇 |
1979年 | 1篇 |
排序方式: 共有938条查询结果,搜索用时 0 毫秒
711.
Sean‐Patrick Riechers Ulf Stahl Christine Lang 《Journal of cellular biochemistry》2009,106(2):327-336
The yeast Saccharomyces cerevisiae is a standard model system to study endocytosis. Here we describe the examination of a representative subset of deletion mutants to identify and locate steps in endocytic transport, endosomal/lysosomal acidification and in intracellular transport of hydrolases in non‐viral transfection processes. When transport in late endocytosis is inhibited, transfection efficiency is significantly enhanced. Similarly, transfection efficiency is enhanced when the pH‐value of the endosomal/vacuolar system is modified. Transfection efficiency is furthermore elevated when the Na+/K+ transport in the endosomal system is disturbed. Finally, we observe enhanced transfection efficiency in mutants disturbed in the CVT/autophagy pathway and in hydrolase transport to the vacuole. In summary, non‐viral transfection efficiency can be significantly increased by either (i) inhibiting the transport of endocytosed material before it enters the vacuole, or (ii) inducing a non‐natural pH‐value of the endosomal/vacuolar system, or (iii) slowing down degradative processes by inhibiting vacuolar hydrolases or the transport between Golgi and late endosome/vacuole. J. Cell. Biochem. 106: 327–336, 2009. © 2008 Wiley‐Liss, Inc. 相似文献
712.
Sonia Gutiérrez-Granados Laura Cervera Amine A. Kamen Francesc Gòdia 《Critical reviews in biotechnology》2018,38(6):918-940
Transient gene expression (TGE) in animal cell cultures has been used for almost 30 years to produce milligrams and grams of recombinant proteins, virus-like particles and viral vectors, mainly for research purposes. The need to increase the amount of product has led to a scale-up of TGE protocols. Moreover, product quality and process reproducibility are also of major importance, especially when TGE is employed for the preparation of clinical lots. This work gives an overview of the different technologies that are available for TGE and how they can be combined, depending on each application. Then, a critical assessment of the challenges of large-scale transient transfection follows, focusing on suspension cell cultures transfected with polyethylenimine (PEI), which is the most widely used methodology for transfection. Finally, emerging opportunities for transient transfection arising from gene therapy, personalized medicine and vaccine development are reviewed. 相似文献
713.
Xiaojie Fan Jingnan Yang Guangyao Wu Meiyi Wang Xiaoxia Cheng Chang Liu Qian Liu Yanan Wen Shuangshuang Meng Zhenxing Wang Xuhong Lin Lei An 《Genetics and molecular biology》2022,45(2)
Transfection efficiency was estimated to optimize the conditions for RNA interference (RNAi), including transfection time, validity, and nucleic acid concentration and type, using the EZ Trans Cell Reagent, a cationic polymer. An shRNA against GFP was designed and transfected into cells using the EZ transfection reagent. The shRNA significantly decreased the expression of GFP. In addition, pre-diluted transfection reagent at room temperature and small nucleic acids increased the transfection efficiency, which peaked at 24 h. Compared with circular nucleic acids, linear nucleic acids showed higher transfection efficiency and a higher genome integration rate. We optimized cationic polymer-mediated RNAi conditions, and our data will be useful for future RNAi studies. 相似文献
714.
激光诱导的基因转染方法研究 总被引:1,自引:0,他引:1
基因转染是研究基因表达、结构和功能的主要实验手段。本文对现有的转染方法进行了分析,并重点探讨了两种激光诱导的基因转染新方法:激光产生冲击波和激光照射纳米微粒的方法,并提出了改进的实验方案。这两种方法都是通过改变细胞质膜通透性实现外源基因的导入,其最大的优点就是对细胞没有损伤。文章的最后说明了基因转染在基因疗法的应用。 相似文献
715.
Jan Oxholm Gordeladze Kjerstin Elisabeth Hvik John Jack Merendino Sylvie Hermouet Silvio Gutkind Domenico Accili 《Journal of cellular biochemistry》1997,64(2):242-257
Previous investigations have demonstrated that both Gs- and the Gi-family of GTP-binding proteins are implicated in differentiation of the 3T3-L1 preadipocyte. In order to further analyze the role of Gsα vs. Gi2α, which are both involved in adenylate cyclase modulation, we transfected undifferentiated 3T3-L1 cells with two sets of G-protein cDNA: the pZEM vector with either wild type, the activating (i.e., GTP-ase inhibiting) R201C-Gsα or the inactivating G226A(H21a)-Gsα point mutations, or the pZIPNeoSV(X) retroviral vector constructs containing the Gi2α wild type or the missense mutations R179E-Gi2α, Q205L-Gi2α, and G204A(H21a)-Gi2α. The activating [R201C]Gsα-mutant did not significantly affect the differentiation process, i.e., increase in the steady-state levels of G-protein subunits, gross appearance, or insulin-elicited deoxy-glucose uptake into 3T3-Ll adipocytes, despite a marked initial increase in hormone-elicited adenylate cyclase activity. The [H21a]Gsα-mutant, on the other hand, enhanced the degree of differentiation slightly, as evidenced by an augmented production of lipid vesicles and insulin-stimulated deoxy-glucose uptake. However, an expected increase in mRNA for hormone-sensitive lipase was not seen. Secondly, it appeared that both activating [R179E]Gi2α or [Q205L]Gi2α mutants reduced cell doubling time in non-confluent 3T3-L1 cell cultures, while [H21a]Gi2α slowed proliferation rate. Furthermore, it seemed that cell proliferation, as evidenced by thymidine incorporation, ceased at a much earlier stage prior to cell confluency when cultures were transfected with the [R179E]Gi2α or [Q205L]Gi2α mutants. Upon differentiation with insulin, dexamethasone, and iBuMeXan, the following cell characteristics emerged: the [R179E]Gi2α and [Q205L]Gi2α mutants consistently enhanced adenylate cyclase activation and cAMP accumulation stimulated by isoproterenol and corticotropin over controls. Deoxy-glucose uptake was also super-activated by the [R179E]Gi2α and [Q205L]Gi2α mutants. Finally, steady-state levels of hormone sensitive lipase mRNA were dramatically increased by [R179E]Gi2α and [Q205L]Gi2α over differentiated controls. The inactivating [H21a]Gi2α-mutant obliterated all signs of preadipocyte differentiation. It is concluded that Gi2 plays a positive and much more important role than Gs in 3T3-L1 preadipocyte differentiation. Cyclic AMP appears to play no role in this process. J. Cell. Biochem. 64:242–257. © 1997 Wiley-Liss, Inc. 相似文献
716.
717.
R. A. Shafei I. A. Zelenina M. L. Semenova V. A. Kolesnikov V. M. Mikhailov V. S. Baranov A. V. Zelenin V. A. Golichenkov 《Russian Journal of Developmental Biology》2000,31(5):326-331
We used the method of particle bombardment (ballistic transfection) to introduce β-galactosidase and human dystrophin genes
into mouse embryos and skeletal muscles of adult mice. We examined the mechanisms of DNA transfer into skeletal muscle cells,
the biological processes accompanying and following this transfer, the susceptibility of various types of muscle cells to
transfection, and the duration of expression of and conditions affecting the introduced genes. We have also developed an effective,
convenient, and practical methods of skeletal muscle transfection. 相似文献
718.
719.
Augmented nuclease activity during cellular senescence in vitro 总被引:1,自引:0,他引:1
The molecular correlates of the limited proliferative potential of normal human diploid fibroblasts and extensive single-strand breaks in the genomic DNA of these cells were examined by transfection analyses in which DNA replication could be uncoupled from DNA damage and repair. Both supercoiled (fmI), and restriction endonuclease-cleaved, linear (fmIII) molecules of a well-defined bacterial plasmid DNA, pBR322, were transfected into, and subsequently recovered from, early and late passage fibroblasts. Southern blot analysis revealed that fmI DNA was converted by random nicks into fmII DNA slightly more rapidly in late passage cells compared with cells at early passage. Similarly, fmII and fmIII DNAs also sustained multiple random nicks and no appreciable net religation of free ends of fmIII DNA could be detected at either passage. In addition, the efficiency of in vitro ligation of fmIII DNA recovered from late passage cells was also reduced, compared with that from early passage cells, as determined by Southern blotting. These data suggest that in the absence of DNA replication, a putative nuclease activity may contribute to DNA damage observed in senescent cells, which, in turn, may be causally related to their limited replicative potential. 相似文献
720.
Asuka Matsui Aya C. Yoshida Mayumi Kubota Masaharu Ogawa Tomomi Shimogori 《Journal of visualized experiments : JoVE》2011,(54)
In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance molecules, local gene transfer or knock- out is required. Gene targeting knock-in or knock-out into local regions is possible to perform with combination with a specific CRE line, which is laborious, costly, and time consuming. Therefore, a simple transfection method, an in utero electroporation technique, which can be performed with short time, will be handy to test the possible function of candidate genes prior to the generation of transgenic animals 1,2. In addition to this, in utero electroporation targets areas of the brain where no specific CRE line exists, and will limit embryonic lethality 3,4. Here, we present a method of in utero electroporation combining two different types of electrodes for simple and convenient gene transfer into target areas of the developing brain. First, a unique holding method of embryos using an optic fiber optic light cable will make small embryos (from E9.5) visible for targeted DNA solution injection into ventricles and needle type electrodes insertion to the targeted brain area 5,6. The patterning of the brain such as cortical area occur at early embryonic stage, therefore, these early electroporation from E9.5 make a big contribution to understand entire area patterning event. Second, the precise shape of a capillary prevents uterine damage by making holes by insertion of the capillary. Furthermore, the precise shape of the needle electrodes are created with tungsten and platinum wire and sharpened using sand paper and insulated with nail polish 7, a method which is described in great detail in this protocol. This unique technique allows transfection of plasmid DNA into restricted areas of the brain and will enable small embryos to be electroporated. This will help to, open a new window for many scientists who are working on cell differentiation, cell migration, axon guidance in very early embryonic stage. Moreover, this technique will allow scientists to transfect plasmid DNA into deep parts of the developing brain such as thalamus and hypothalamus, where not many region-specific CRE lines exist for gain of function (GOF) or loss of function (LOF) analyses. 相似文献