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1.
Wolf Bertling 《Bioscience reports》1987,7(2):107-112
We used empty capsids ofpolyoma virus to transfer DNA fragments and DNA/protein complexes into human cells. We encapsulated labeled and unlabeled single stranded DNA fragments by viral capsids. A complex of DNA with a DNA binding protein, recA, will also be taken up by the capsids, whereas the free protein is not incorporated. We further compared this gentle biological method of DNA transfection with a well-established physical method, electroporation. Electroporation also allows the transfer of DNA as well as protein into cells, although there is no proof that a DNA/protein complex can survive the procedure functionally. Whereas the viability of capsid transfected cells is unaffected (100%), electroporation reduces the viability to 90–95%. On the other hand, the amount of DNA found in the nucleus of electroporated cells is higher than for cells treated with loaded viral capsids. 相似文献
2.
We found previously that neural crest cells in turtle embryos migrated into the lung buds and melanocytes were located in the lungs. The finding suggested to us that the lungs provide a stimulatory factor(s) to the differentiation of neural crest cells into melanocytes. We have established lung cell lines to facilitate analysis of the interactions of neural crest cells with the environment in melanocyte development. One cell line, TLC-2, was found to produce a putative melanization-stimulating activity (MSA), which promoted the melanocyte differentiation in vitro of avian neural crest cells. The TLC-2-derived MSA was different from that of basic fibroblast growth factor (bFGF), α-melanocyte stimulating hormone (α-MSH), and steel factor (SLF). Its molecular weight was estimated to be within the range of 150 kD. Our findings suggest that MSA may be a novel factor exercising a positive control over melanocyte differentiation. 相似文献
3.
Sheri L. Holmen Matt W. Vanbrocklin Robert R. Eversole Susan R. Stapleton Leonard C. Ginsberg 《In vitro cellular & developmental biology. Animal》1995,31(5):347-351
Cationic lipids are an effective means for transfecting nucleic acids into a variety of cell types. Very few of these lipids,
however, have been reported to be effective with primary cells. We report on the efficacy of several commercially available
cationic lipid reagents to transfect plasmid DNA into primary rat hepatocytes in culture. The reagents tested in this study
include TransfectAce, LipofectAmine, Lipofectin, N-[1-(2,3-dioleyloxy)propyl]-n,n,n-trimethylammoniumchloride (DOTMA), (N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethyl-ammonium
methylsulfate (DOTAP), and cetyltrimethyl-ammonium bromide/dioleoylphosphatidylethanol-amine (CTAB/DOPE). Electron micrographic
(EM) studies indicate that similar size Lipofectin and DOTAP vesicles contain DNA-like material internally and that these
vesicles attach to the cell membrane. DOTAP vesicles are multilamellar, appear as clusters, and have a high DNA-to-lipid ratio.
Lipofectin vesicles appear to attach to the cell surface as individual vesicles. The EM observations are consistent with current
theories on the mechanism of transfection by cationic lipids. While Lipofectin has proven to be effective in transfection
studies of primary cells in culture, we have found DOTAP to be a viable alternative. DOTAP yields transfection rates in hepatocytes
comparable to DOTMA and Lipofectin, however, at lower concentrations of reagent and at considerably less cost. Optimal conditions
for transfecting 5 μg of plasmid DNA with DOTAP were achieved by utilizing multilamellar (vortexed) vesicles at a concentration
of 15 μg DOTAP per 2 ml media in 60-mm plates for 2 h transfection time. In this study, DOTAP has proven to be economical,
easy to prepare, and very effective in transfecting DNA into primary rat hepatocytes. 相似文献
4.
Delivery of DNA into mammalian cells by receptor-mediated endocytosis and gene therapy 总被引:8,自引:0,他引:8
The correction of genetically based disorders by the introduction of a therapeutic genetic construct into the appropriate
cell type (“gene therapy”), has become a distinct possibility in recent years. In order for gene therapy to be a practical
alternative to more conventional pharmaceutical approaches to treatment, it must be administrable in vivo. This demands that
a system be developed that can specifically target the DNA to the desired cell type once introduced into the patient. Among
the procedures that are currently being pursued, the delivery of DNA to cells by receptor mediated endocytosis (RME), comes
closest to fulfilling this crucial requirement.
The natural physiological process of RME can be exploited to deliver genetic material to cells. An antibody or ligand to a
cell surface receptor that is known to undergo endocytosis, is complexed with DNA through a covalently linked polycationic
adjunct (e.g., polylysine, protamines). Such complexes retain their binding specificity to the cell surface and are taken
up into the cell where they enter the endosomal compartment via normal endocytotic processes. In addition, steps must be taken
to avoid degradation of the DNA within the endosome-lysosome. Cells can be treated with the lysosomatropic agent chloroquine
during the transfection procedure. Alternatively, the components of viruses that enter cells by endocysis and possess an endosomal
“break out” capacity can be used. Replication defective adenovirus coupled to the ligand-DNA complex gives transfection efficiencies
of virtually 100% on tissue culture cells in vitro. Synthetic peptides that mimic the membrane fusing region of influenza
virus hemagglutinin, have also been successfully used as part of the ligand-DNA complex to bring about endosomal escape.
Preliminary studies have demonstrated the potential of this method to specifically target DNA to the cell type of choice in
vivo. Delivery of genes by receptor-mediated endocytosis offers the greatest hope that gene therapy can be an inexpensive,
easily applicable, widespread technology. 相似文献
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In the fowl the primordial germ cells accumulate in the germinal crescent to the anterior of the two-day embryo. A simple ballistic device has been used to fire tungsten particles (mean diameter 1.5 m) into this region. By coating these projectiles with vector DNA it is possible to transfect these cells. Hatchlings produced by this technique were raised to sexual maturity and shown to contain the foreign DNA in their sperm. G1 offspring containing this DNA were also produced in roughly 20% of these cockerels. In the majority of cases the vector DNA disappeared from the G1 generation as they matured suggesting that in these cases it had been transmitted episomally. 相似文献