- High siRNA transfection efficiency
- Functional gene silencing post siRNA delivery
- Compatibility with diverse growth conditions (with and without serum)
- Low cytotoxicity
- Easy-to-use protocols for both adherent and suspension cells
Contents
- Hydrated GeneSilencer Lipid
- siRNA Diluent (corresponding ratio for each kit)
Storage
GeneSilencer siRNA Transfection Reagent is shipped at room temperature, as similar to other gene therapy products. For maximum stability, store all reagents at 4篊 upon receipt. If stored properly, all components are stable for 6 months.GeneSilencer Transfection Reagent Kit
- 50 rxn kit, 0.18 ml, T500020
- 200 rxn kit, 0.75 ml, T500750
- 1000 rxn kit, 5 x 0.75 ml, T505750
- 10 rxn sample kit, 0.03 ml, T500020S
Long Noncoding RNA pancEts-1 Promotes Neuroblastoma Progression through hnRNPK-Mediated β-Catenin Stabilization.Li, D.,et al (2018) Cancer Res. 78 (5): 1169-1183. Link
Apoptotic cells trigger the ABCA1/STAT6 pathway leading to PPAR‐γ expression and activation in macrophages.Kim, M-J.,et al (2018) J. Leukocyte Biol. 103(5): 885-895. Link
Transient Silencing of DNA Repair Genes Improves Targeted Gene Integration in the Filamentous Fungus Trichoderma reesei.Chum, P.Y.,et al ((2017) Appl. Environ. Microbiol. 83(15): e00535-17. Link
Programming of macrophages by apoptotic cancer cells inhibits cancer progression through exosomal PTEN and PPARγ ligands.Kim, Y-B.,et al (2017) bioRxiv 217562: 1-75. Link
Role of Sphingosine Kinase 1 and S1P Transporter Spns2 in HGF-mediated Lamellipodia Formation in Lung Endothelium.Fu, P.,et al (2016) J. Biol. Chem. 291(53): 27187?27203. Link
Ubiquitin-specific Protease 20 Regulates the Reciprocal Functions of β-Arrestin2 in Toll-like Receptor 4-promoted Nuclear Factor κB (NFκB) Activation.Jean-Charles, P-Y., (2016) J. Biol. Chem. 291 (14): 7450 ?7464. Link
Tolerogenic nanoparticles inhibit T cell杕ediated autoimmunity through SOCS2.Yeste, A.,et al (2016) Sci. Signal. 9(433): RA61. Link
Synthesis of an Endogenous Steroidal Na Pump Inhibitor Marinobufagenin, Implicated in Human Cardiovascular Diseases, Is Initiated by CYP27A1 via Bile Acid Pathway.Fedorova, O.V.,et al (2015) Circ Cardiovasc Genet. 8(5): 736-45. Link
Thioredoxin Activates MKK4-NFκB Pathway in a Redox-dependent Manner to Control Manganese Superoxide Dismutase Gene Expression in Endothelial Cells.Kundumani-Sridharan, V.,et al (2015) J. Biol. Chem. 290(28): 17505?17519. Link
Identification, Mechanism of Action, and Antitumor Activity of a Small Molecule Inhibitor of Hippo, TGF-β, and Wnt Signaling Pathways.Basu, D.,et al (2014) Mol. Cancer Ther. 13(6): 1457-1467. Link
PKC Potentiates Tyrosine Kinase Inhibitors STI571 and Dasatinib Cytotoxic Effect.Tob蚾, A.,et al (2014) Anticancer Res. 34(7): 3347-3356. Link
TREK2 Expressed Selectively in IB4-Binding C-Fiber Nociceptors Hyperpolarizes Their Membrane Potentials and Limits Spontaneous Pain.Acosta, C.,et al (2014) J. Neurosci. 34(4): 1494-1509. Link
RhoA/Phosphatidylinositol 3-Kinase/Protein Kinase B/Mitogen-Activated Protein Kinase Signaling after Growth Arrest朣pecific Protein 6/Mer Receptor Tyrosine Kinase Engagement Promotes Epithelial Cell Growth and Wound Repair via Upregulation of Hepatocyte Growth Factor in Macrophages.Lee, Y-J.,et al (2014) J. Pharma. Exp. Thera. 350(3): 563-577. Link
GeneSilencer Cell Line Citations: 3T3-L1, A549, A7, AGS, ASM Transformed, B16-F1, BEL7404, Bronchial C166, C2C12, C6R, CHO-K1, DC2.4, GS-KB-3-1, H22, H441, H460, H9c2, hCMEC/D3, HCP40, HCT-116, HEK 293, HEK 293T, HEK-293, HeLa, HepG2, HL-60, HMEC-1, HPAEC, HT29, HUT-78, JAR, Jurkat, KATOIII, L/Stab-2, LNCaP, M2, McA-RH 7777, MCF-7, MDA MB-231, MDA MB-468, MDA-MB-435, MG63, MKN 28, MKN 45, MV-4-11, Neuro2a, NIH-3T3, OK, PC-12, Cortical Neurons, Hippocampal Neurons, Macrophage, Pulmonary Artery, Endothelium, Dendritic Cells(Bone Marrow), Cortical Neurons, Coronary Artery Endothelial, Peritoneal Macrophage, Dorsal Root Ganglion Neurons, Retinal Ganglion, Dorsal Root Ganglion, Schwann Cells Cardiac Fibroblast, Cardiac Myocytes (Neonatal), Lung Microvascular Endothelial Cells, Aortic Muscle Cells, Dendritic Cells, Cerebellar Neurons, Osteoblast, HMVEC, Cardiac Myocytes, T-Cells (CD3+), Cortical Neurons, Dendritic Cells, Cortical Neurons, Aveolar Type II Cells, Macrophage, Dorsal Root Ganglion Lacrimal Gland Acinar Cells, Bone Marrow, Dendritic Cells, Pulmonary Artery Endothelium, Primary HPAEC. RAW 264.7, Renca, SaOs2, Saos-2, SCCH196, SNB19, SW480, U20S
Figure Legend: HEK 293 cells were transfected with 5, 10, 30 or 50 nM of Silencer FAM Labeled GAPDH siRNA with either GeneSilencer (Genlantis) or siPORT NeoFX (Ambion) siRNA transfection reagents according to the manufacturer"s recommended protocols. Cells were incubated for 48 hours then fixed, permeablized, and incubated with AlexaFluor 555 phalloidin (Invitrogen), which stains cellular actin red. Cells were then mounted on slides and stained with DAPI (Invitrogen), which stains the cell nuclei blue. Transfected cells were visualized by fluorescence microscopy using identical exposure times for FITC, TRITC, and DAPI. The transfected GAPDH siRNA is localized in the cell cytosol and can be seen as green fluorescent specks or dots.
HeLa S3 Cells (click on images to enlarge)
Figure Legend: HeLa S3 cells were transfected with 5, 10, 30 or 50 nM of Silencer FAM labeled GAPDH siRNA with either GeneSilencer (Genlantis) or siPORT&tm; NeoFX&tm; (Ambion) siRNA transfection reagents according to the manufacturer"s recommended protocols. Cells were incubated for 48 hours then fixed, permeablized, and incubated with AlexaFluor 555 phalloidin (Invitrogen), which stains cellular actin red. Cells were then mounted on slides and stained with DAPI (Invitrogen), which stains the cell nuclei blue. Transfected cells were visualized by fluorescence microscopy using identical exposure times for FITC, TRITC, and DAPI. The transfected GAPDH siRNA is localized in the cell cytosol and can be seen as green fluorescent specks or dots.
How to Design siRNA (Small Interfering RNA)
The sequence of the siRNA can be selected as follows:
The sequence selection process has no other constraints. It is important to note that structure within thetargeted mRNA appears to have minimal effect on the availability of the mRNA target and efficacy of thesiRNA silencing approach. To-date, successful silencing has been achieved using the above method toselect the target sequence, although the method is essentially random with respect to accounting for mRNAstructure.Although siRNA silencing appears to be extremely effective by selecting a single target in the mRNA, itmay be desirable to design and employ two independent siRNA duplexes to control for specificity of thesilencing effect. This recommendation is only for specificity for it is yet unknown if the targeting of a geneby two different siRNA duplexes would be more effective than using a single siRNA duplex. It is believedthat the rate-limiting component of the siRNA effect is the availability of cellular nuclease components andnot mRNA target availability. Therefore, doubling the number of siRNA duplexes is not expected to doublethe rate or efficiency of silencing.If the selected siRNA duplex(es) do not function for silencing, the following steps are recommended. First,a search is conducted for sequencing errors in the gene and possible polymorphisms. Initial studies on thespecificity of target recognition by siRNA duplexes indicates that a single point mutation located in thepaired region of an siRNA duplex is sufficient to abolish target mRNA degradation. Second, a reexaminationis performed to confirm whether the cell line is from the expected species. Third, a secondand/or third target are selected and the corresponding siRNA duplexes prepared.
Cell Line | Cell Type | Source | Genlantis Product | Citation |
3T3-L1 | Embryo | Swiss Mouse | GeneSilencer siRNA Transfection Reagent | Mizuarai, S., Miki, S., Araki, H., Takahashi, K. and Kotani, H.(2005) Identification of Dicarboxylate Carrier Slc25a10 as Malate Transporter in de Novo Fatty Acid Synthesis. J. Biol. Chem.280(37): p. 32434-32441. |
A549 | Lung Carcinoma | Human | GeneSilencer siRNA Transfection Reagent | Ito, K., Yamamura, S., Essilfie-Quaye, S., Cosio, B., Ito, M., Barnes, P.J. and Adcock, I.M. (2006) Histone deacetylase 2-mediated deacetylation of the glucocorticoid receptor enables NFκB suppression. J. Exp. Med. 203(1): 7-13. |
A549 | Lung Carcinoma | Human | GeneSilencer siRNA Transfection Reagent | Stepulak, A., Sifringer, M., Rzeski, W., Endesfelder, S., Gratopp, A., Pohl, E.E., Bittigau, P., Hansen, H.H., Stryjecka-Zimmer, M., Turski, L. and Ikonomidou, C. (2005) NMDA antagonist inhibits the extracellular signal-regulated kinase pathway and suppresses cancer growth. Proc. Natl. Acad. Sci. 102: 15605 - 15610. |
A7 | Melanoma | Human | GeneSilencer siRNA Transfection Reagent | Scott, M.G.H., Pierotti, V., Storez, H., Lindberg, E., Thuret, A., Muntaner, O., Labbe-Jullie, C., Pitcher, J.A. and Marullo, S. (2006) Cooperative Regulation of Extracellular Signal-Regulated Kinase Activation and Cell Shape Change by Filamin A and β-Arrestins. Mol. & Cell Biol, 26: 3432-3445. |
AGS | Gastric Epithelium | Human | GeneSilencer siRNA Transfection Reagent | Nagasako, T., Sugiyama, T., Mizushima, T., Miura, Y., Kato, M. and Asaka, M. (2003) Up-regulated Smad5 Mediates Apoptosis of Gastric Epithelial Cells Induced by Helicobacter pylori Infection. J. Biol. Chem.: 278: 4821 - 4825. |
AGS | Gastric Epithelium | Human | GeneSilencer siRNA Transfection Reagent | Varro, A., Noble, P-J. M., Pritchard, D.M., Kennedy, S., Hart, C.A., Dimaline, R. and Dockray, G.J. (2004) Helicobacter pylori Induces Plasminogen Activator Inhibitor 2 in Gastric Epithelial Cells through Nuclear Factor-B and RhoA: Implications for Invasion and Apoptosis Cancer Res. 64: 1695 - 1702. |
ASM Transformed | Airway Smooth Muscle Cells | Human | GeneSilencer siRNA Transfection Reagent | Tran, T., Ens-Blackie, K., Rector, E.S., Stelmack, G.L., McNeill, K.D., Tarone, G., Gerthoffer, W.T., Unruh, H. and Halayko, A.J. (2007) Laminin-binding Integrin α7 is Required for Contractile Phenotype Expression by Human Airway Myocyte. Am. J. Respir. Cell Mol. Biol. 37(6): 668-80. |
B16-F1 | Melanoma | Mouse | GeneSilencer siRNA Transfection Reagent | Bertling, E., Hotulainen, P., Mattila, P.K., Matilainen, T., Salminen, M., and Lappalainen, P. (2004) Cyclase-associated-protein 1 (CAP1) promotes cofilin-induced actin dynamics in mammalian nonmuscle cells. Mol. Biol. Cell 15 (5): p. 2324-2334. |
B16-F1 | Melanoma | Mouse | GeneSilencer siRNA Transfection Reagent | Hotulainen, P., Paunola, E., Vartiainen, M.K. and Lappalainen, P. (2005) Actin-depolymerizing Factor and Cofilin-1 Play Overlapping Roles in Promoting Rapid F-Actin Depolymerization in Mammalian Nonmuscle Cells. Mol. Biol. Cell. 16(2): p. 649-664. |
BEL7404 | Hepatoma | Human | GeneSilencer siRNA Transfection Reagent | Liang, X-J, Finkel, T., Shen, D-W, Yin, J-J, Aszalos, A. and Gottesman, M.M. (2008) SIRT1 Contributes in Part to Cisplatin Resistance in Cancer Cells by Altering Mitochondrial Metabolism. Mol Cancer Res 6(9): 1499-506. |
Bronchial | Smooth Muscle | Human | GeneSilencer siRNA Transfection Reagent | Nunes, R.O., Schmidt, M., Dueck, G., Baarsma, H., Halayko, A.J., Kerstjens, H.A.M., Meurs, H. and Gosens, R. (2008) GSK-3/β-catenin signaling axis in airway smooth muscle: role in mitogenic signaling Am J Physiol Lung Cell Mol Physiol. 294: L1110 - L1118. |
C166 | Yolk Sack Endothelium | Mouse | GeneSilencer siRNA Transfection Reagent | Zhou, X., Stuart, A., Dettin, L.E., Rodriguez, G., Hoel, B. and Gallicano G.I. (2004) Desmoplakin is required for microvascular tube formation in culture. J. Cell Sci. 117: 3129-3140. |
C2C12 | Myoblast | Mouse | GeneSilencer siRNA Transfection Reagent | Evangelisti, C., Tazzari, P.L., Riccio, M., Fiume, R., Hozumi, Y., Fala, F., Goto, K.., Manzoli, L., Cocco, L. and Martelli, A.M. (2007) Nuclear diacylglycerol kinase-ζ is a negative regulator of cell cycle progression in C2C12 mouse myoblasts. FASEB J. 21: 3297 - 3307 |
C6R | Glioma | Rat | GeneSilencer siRNA Transfection Reagent | Saarikangas, J., Hakanen, J., Mattila, P.K., Grumet, M., Salminen, M. and Lappalainen, P. (2008) ABBA regulates plasma-membrane and actin dynamics to promote radial glia extension. J Cell Sci. 121(Pt 9):1444-54. |
CHO-K1 | Ovary | Chinese Hamster | GeneSilencer siRNA Transfection Reagent | Kheifets, V., Bright, R., Inagaki, K., Schechtman, D. and Mochly-Rosen, D. (2006) Protein Kinase C δ PKC-Annexin V Interaction: a required step in δ PKC translocation and function. J. Biol. Chem. 281(32): p. 23218-23226. |
DC2.4 | Bone Marrow | Mouse | GeneSilencer siRNA Transfection Reagent | Jing, H., Yen, J-H and Ganea, D. (2004) A novel signaling pathway mediates the inhibition of CCL3/4 expression by PGE2. J. Biol. Chem. 279(53): p. 55176-55186. |
GS-KB-3-1 | Epidermoid Adenocarcinoma | Human | GeneSilencer siRNA Transfection Reagent | Liang, X-J, Finkel, T., Shen, D-W, Yin, J-J, Aszalos, A. and Gottesman, M.M. (2008) SIRT1 Contributes in Part to Cisplatin Resistance in Cancer Cells by Altering Mitochondrial Metabolism. Mol Cancer Res 6(9): 1499-506. |
H22 | Hepatoma | Mouse | GeneSilencer siRNA Transfection Reagent | Huang, B., Lei, Z., Zhang, G-M, Li, D., Song, C., Li, B., Liu, Y., Yuan, Y., Unkeless, J., Xiong, H. and Feng, Z-H (2008) SCF-mediated mast cell infiltration and activation exacerbate the inflammation and immunosuppression in tumor microenvironment. Blood 112(4): 1269-79. |
H22 | Hepatoma | Mouse | GeneSilencer siRNA Transfection Reagent | Huang, B., Zhao, J., Shen, S., Li, H., He, K-L, Shen, G-X, Mayer, L., Unkeless, J., Li, D., Yuan, Y., Zhang, G.-M., Xiong, H. and Feng, Z.-H. (2007) Listeria monocytogenes Promotes Tumor Growth via Tumor Cell Toll-Like Receptor 2 Signaling. Cancer Res. 67(9): p. 4346-4352. |
H441 | Lung Adenocarcinoma | Human | GeneSilencer siRNA Transfection Reagent | Zhang, Y-A., Nemunaitis, J., Samuel, S.K., Chen, P., Shen, Y. and Tong, A.W. (2006) Antitumor Activity of an Oncolytic Adenovirus-Delivered Oncogene Small Interfering RNA. Cancer Res. 66(19): 9736-9743. |
H460 | Lung Cancer | Human | GeneSilencer siRNA Transfection Reagent | Ren, J., Shi, M., Liu, R., Yang, Q-H., Johnson, T., Skarnes, W.C. and Du, C. (2005) The Birc6 (Bruce) gene regulates p53 and the mitochondrial pathway of apoptosis and is essential for mouse embryonic development. Proc. Natl. Acad. Sci. USA. 102(3): p. 565-570. |
H9c2 | Myoblast | Rat | GeneSilencer siRNA Transfection Reagent | Saotome, M., Safiulina, D., Szabadkai, G., Das, S., Fransson, A., Aspenstrom, P., Rizzuto, R. and Hajnoczky, G. (2008) Bidirectional Ca2+-dependent control of mitochondrial dynamics by the Miro GTPase. Proc Natl Acad Sci U S A 105(52): p. 20728. |
hCMEC/D3 | Brain Microvascular Endothelia | Human | GeneSilencer siRNA Transfection Reagent | Huang, W., Eum, S.Y., Andras, I.E., Hennig, B. and Toborek, M. (2009) PPAR{alpha} and PPAR{gamma} attenuate HIV-induced dysregulation of tight junction proteins by modulations of matrix metalloproteinase and proteasome activities. FASEB J 13 Jan 2009. |
HCP40 | Colon adenocarcinoma | null | GeneSilencer siRNA Transfection Reagent | Yao, K., Shida, S., Selvakumaran, M., Zimmerman, R., Simon, E., Schick, J., Haas, N.B., Balke, M., Ross, H., Johnson, S.W. and O"Dwyer, P.J. (2005) Macrophage Migration Inhibitory Factor Is a Determinant of Hypoxia-Induced Apoptosis in Colon Cancer Cell Lines. Clin Cancer Res. 11 (20): 7264 - 7272. |
HCT-116 | Colon Carcinoma | Human | GeneSilencer siRNA Transfection Reagent | Wang, J., Rajput, A., Kan, J.L.C., Rose, R., Liu, X., Kuropatwinski, K., Hauser, J., Beko, A., Dominquez, I., Sharratt, E.A., Brattain, L., LeVea, C., Sun, F., Keane, D.M., Gibson, N.W. and Michael G. Brattain (2009) Knockdown of Ron kinase inhibits mutant PI3 kinase and reduces metastasis in human colon carcinoma. J. Biol. Chem. 284 (16) 10912-10922. |
HCT-116 | Colon Adenocarcinoma | Human | GeneSilencer siRNA Transfection Reagent | Yao, K., Shida, S., Selvakumaran, M., Zimmerman, R., Simon, E., Schick, J., Haas, N.B. Balke, M., Ross, H., Johnson, S.W. and O"Dwyer, P.J. (2005) Macrophage Migration Inhibitory Factor Is a Determinant of Hypoxia-Induced Apoptosis in Colon Cancer Cell Lines. Clin. Can. Res. 11: 7264-7272. |
HEK 293 | Embryonic Kidney | Human | GeneSilencer siRNA Transfection Reagent | Ahn, S., Nelson, C.D., Garrison, T.R., Miller, W.E. and Lefkowitz, R.J. (2003) Desensitization, internalization, and signaling functions of -arrestins demonstrated by RNA interference. Proc. Natl. Acad. Sci. 100: 1740 - 1744. |
HEK 293 | Embryonic Kidney | Human | GeneSilencer siRNA Transfection Reagent | Ahn, S., Shenoy, S.K., Wei, H. and Lefkowitz, R.J. (2004) Differential Kinetic and Spatial Patterns of {beta}-Arrestin and G Protein-mediated ERK Activation by the Angiotensin II Receptor. J. Biol. Chem. 279 (34): 35518-35525. |
HEK 293 | Embryonic Kidney | Human | GeneSilencer siRNA Transfection Reagent | Ahn, S., Wei, H., Garrison, T.R. and Lefkowitz, R.J. (2004) Reciprocal Regulation of Angiotensin Receptor-activated Extracellular Signal-regulated Kinases by (beta)-Arrestins 1 and 2. J. Biol. Chem. 279: 7807-7811. |
HEK 293 | Embryonic Kidney | Human | GeneSilencer siRNA Transfection Reagent | Khundmiri, S.J., Dean, W.L., McLeish, K.R. and Lederer, E.D. (2005) Parathyroid Hormone-mediated Regulation of Na+-K+-ATPase Requires ERK-dependent Translocation of Protein Kinase C{alpha}. J. Biol. Chem. 280(10): p. 8705-8713. |
HEK 293 | Embryonic Kidney | Human | GeneSilencer siRNA Transfection Reagent | Kim, J., Ahn, S., Ren, X-R., Whalen, E.J., Reiter, E., Wei, H. and Lefkowitz, R.J. (2005) Functional antagonism of different G protein-coupled receptor kinases for {beta}-arrestin-mediated angiotensin II receptor signaling. Proc. Natl. Acad. Sci.驴102: 1442 - 1447. |
HEK 293 | Embryonic Kidney | Human | GeneSilencer siRNA Transfection Reagent | Kohout, T.A., Nicholas, S.L., Perry, S.J., Reinhart, G., Junger, S. and Struthers, R. (2004) Differential Desensitization, Receptor Phosphorylation, {beta}-Arrestin Recruitment, and ERK1/2 Activation by the Two Endogenous Ligands for the CC Chemokine Receptor 7. J. Biol. Chem. 279 (22): p. 23214-23222. |
HEK 293 | Embryonic Kidney | Human | GeneSilencer siRNA Transfection Reagent | Liang, Y., Yu, W., Li, Y., Yan, X., Huang, Q. and Zhu, X. (2004) Nudel functions in membrane traffic mainly through association with Lis1 and cytoplasmic dynein. J. Cell Biol. 164 (4): 557-566. |
HEK 293 | Embryonic Kidney | Human | GeneSilencer siRNA Transfection Reagent | Ren, X-R., Reiter, E., Ahn, S., Kim, J., Chen, W. and Lefkowitz, R.J. (2005) Different G protein-coupled receptor kinases govern G protein and {beta}-arrestin-mediated signaling of V2 vasopressin receptor. Proc. Natl. Acad. Sci. 102: 1448 - 1453. |
HEK 293 | Embryonic Kidney | Human | GeneSilencer siRNA Transfection Reagent | Shenoy, S.K., Drake, M.T., Nelson, C.D., Houtz, D.A., Xiao, K., Madabushi, S., Reiter, E., Premont, R.T., Lichtarge, O. and Lefkowitz, R.J. (2006) {beta}-Arrestin-dependent, G Protein-independent ERK1/2 Activation by the {beta}2 Adrenergic Receptor. J. Biol. Chem. 281(2): 1261-1273. |
HEK 293 | Embryonic Kidney | Human | GeneSilencer siRNA Transfection Reagent | Shikama, Y., Yamada, M., Miyashita, T. (2004) Caspase-8 and caspase-10 activate NF-B through RIP, NIK and IKK kinases. Eur. J. Immun. 33 (7): 1998-2006. |
HEK 293 | Embryonic Kidney | Human | GeneSilencer siRNA Transfection Reagent | Wei, H., Ahn, S., Barnes, W.G. and Lefkowitz, R.J., (2004) Stable interaction between beta -arrestin2 and AT 1A receptor is required for beta -arrestin2 mediated activation of extracellular signal-regulated kinase 1 and 2. J. Biol. Chem. 279: 48255-48261. |
HEK 293 | Embryonic Kidney | Human | GeneSilencer siRNA Transfection Reagent | Wei, H., Ahn, S., Shenoy, S.K., Karnik, S.S., Hunyady, L., Luttrell, L.M. and Lefkowitz, R.J. (2003) Independent -arrestin 2 and G protein-mediated pathways for angiotensin II activation of extracellular signal-regulated kinases 1 and 2. Proc. Natl. Acad. Sci. 100: 10782 - 10787. |
HEK 293 | Embryonic Kidney | Human | GeneSilencer siRNA Transfection Reagent | Wu, J-H, Goswami, R., Kim, L.K., Miller, W.E., Peppel, K. and Freedman, N.J. (2005) The Platelet-derived Growth Factor Receptor-{beta} Phosphorylates and Activates G Protein-coupled Receptor Kinase-2: a Mechanism for Feedback Inhibition. J. Biol. Chem. 280(35): p. 31027-31035. |
HEK 293T | Embryonic Kidney | Human | GeneSilencer siRNA Transfection Reagent | Wei, H., Ahn, S., Shenoy, S.K., Karnik, S.S., Hunyady, L., Luttrell, L.M. and Lefkowitz, R.J. (2003) Independent -arrestin 2 and G protein-mediated pathways for angiotensin II activation of extracellular signal-regulated kinases 1 and 2. Proc. Natl. Acad. Sci. 100: 10782 - 10787. |
HEK-293 | Embryonic Kidney | Human | GeneSilencer siRNA Transfection Reagent | Ahn, S., Kim, J., Hara, M.R., Ren, X. and Lefkowitz, R.J. (2009) β -arrestin2 mediates anti-apoptotic signaling through regulation of bad phosphorylation. J Biol Chem 284(13): 8855-65. |
HEK-293 | Embryonic Fibroblast | Human | GeneSilencer siRNA Transfection Reagent | Barthet, G., Framery, B., Gaven, F., Pellissier, L., Reiter, E., Claeysen, S., Bockaert, J. and Dumuis, A. (2007) 5-Hydroxytryptamine 4 Receptor Activation of the Extracellular Signal-regulated Kinase Pathway Depends on Src Activation but Not on G Protein or beta-Arrestin Signaling. Mol. Biol. Cell. 18(6): 1979-1991. |
HEk-293 | Embryonic Kidney | Human | GeneSilencer siRNA Transfection Reagent | El-Shewy, H.M., Johnson, K.R., Lee, M-H, Jaffa, A.A., Obeid, L.M. and Luttrell, L.M. (2006) Insulin-like Growth Factors Mediate Heterotrimeric G Protein-dependent ERK1/2 Activation by Transactivating Sphingosine 1-Phosphate Receptors . J. Biol. Chem. 281(42): 31399-31407. |
HEK-293 | Embryonic Kidney | Human | GeneSilencer siRNA Transfection Reagent | El-Shewy, H.M., Lee, M-H., Obeid, L.M., Jaffa, A.A. and Luttrell, L.M. (2007) The Insulin-like Growth Factor Type 1 and Insulin-like Growth Factor Type 2/Mannose-6-phosphate Receptors Independently Regulate ERK1/2 Activity in HEK293 Cells. J. Biol. Chem. 282(36): 26150-26157. |
HEK-293 | Embryonic Kidney | Human | GeneSilencer siRNA Transfection Reagent | Gesty-Palmer, D., Chen, M., Reiter, E., Ahn, S., Nelson, C.D., Wang, S., Eckhardt, A.E., Cowan, C.L., Spurney, R.F., Luttrell, L.M. and Lefkowitz, R.J. (2006) Distinct beta-Arrestin- and G Protein-dependent Pathways for Parathyroid Hormone Receptor-stimulated ERK1/2 Activation. J. Biol. Chem. 281(16): 10856-10864. |
HEK-293 | Embryonic Kidney | Human | GeneSilencer siRNA Transfection Reagent | IM Kim, DG Tilley, J Chen, NC Salazar, EJ Whalen, JD Violin, and HA Rockman (2008) Beta-blockers alprenolol and carvedilol stimulate beta-arrestin-mediated EGFR transactivation. Proc Natl Acad Sci 105(38): 14555-60. |
HEK-293 | Embryonic Kidney | Human | GeneSilencer siRNA Transfection Reagent | Kani, S., Nakayama, E., Yoda, A., Onishi, N., Sougawa, N., Hazaka, Y., Umeda, T., Takeda, K., Ichijo, H., Hamada, Y. and Minami, Y. (2007) Chk2 kinase is required for methylglyoxal-induced G2/M cell-cycle checkpoint arrest: implication of cell-cycle checkpoint regulation in diabetic oxidative stress signaling. Genes Cells. 12(8): 919-928. |
HEk-293 | Embryonic Kidney | Human | GeneSilencer siRNA Transfection Reagent | Kara, E., Crepieux, P., Gauthier, C., Martinat, N., Piketty, V., Guillou, F., and Reiter, E. (2006) A Phosphorylation Cluster of Five Serine and Threonine Residues in the C-Terminus of the Follicle-Stimulating Hormone Receptor Is Important for Desensitization But Not for β-Arrestin-Mediated ERK Activation. Mol. Endocrinol. 20(11): p. 3014-3026. |
HEK-293 | Embryonic Kidney | Human | GeneSilencer siRNA Transfection Reagent | Nelson, C.D., Kovacs, J.J., Nobles, K.N., Whalen, E.J. and Lefkowitz, R.J. (2008) β-Arrestin Scaffolding of Phosphatidylinositol 4-Phosphate 5-Kinase Iα Promotes Agonist-stimulated Sequestration of the {beta}2-Adrenergic Receptor. J. Biol. Chem. 283(30): p. 21093-21101. |
HEK-293 | Embryonic Kidney | Human | GeneSilencer siRNA Transfection Reagent | Noma, T., Lemaire, A., Prasad, S.V.N., Barki-Harrington, L., Tilley, D.G., Chen, J., Le Corvoisier, P., Violin, J.D., Wei, H., Lefkowitz, R.J. and Rockman, H.A. (2007) β-Arrestin mediated β1-adrenergic receptor transactivation of the EGFR confers cardioprotection. J. Clin. Invest. 117(9): p. 2445-2458. |
HEk-293 | Embryonic Kidney | Human | GeneSilencer siRNA Transfection Reagent | Rajagopal, K., Whalen, E.J., Violin, J.D., Stiber, J.A., Rosenberg, P.B., Premont, R.T., Coffman, T.M., Rockman, H.A. and Lefkowitz, R.J. (2006) β-Arrestin2-mediated inotropic effects of the angiotensin II type 1A receptor in isolated cardiac myocytes. PNAS. 103: 16284 - 16289. |
HEK-293 | Embryonic Kidney | Human | GeneSilencer siRNA Transfection Reagent | Shenoy, S.K., Xiao, K., Venkataramanan, V., Snyder, P.M., Freedman, N.J. and Weissman, A.M. (2008) NEDD4 mediates agonist-dependent ubiquitination, lysosomal targeting and degradation of the beta 2 adrenergic receptor. J. Biol. Chem. 283: 22166 - 22176. |
HEK-293 | Embryonic Kidney | Human | GeneSilencer siRNA Transfection Reagent | Violin, J.D., DeWire, S.M., Barnes, W.G. and Lefkowitz, R.J. (2006) G Protein-Coupled Receptor Kinase and β-arrestin mediated desensitization of the angiotensin II type 1A receptor elucidated by diacylgylcerol dynamics. J. Biol. Chem. 281: 36411 - 36419. |
HeLa | Cervical Carcinoma | Human | GeneSilencer siRNA Transfection Reagent | Bentley, A.M., Normand, G., Hoyt, J. and King, R.W. (2007) Distinct Sequence Elements of Cyclin B1 Promote Localization to Chromatin, Centrosomes, and Kinetochores during Mitosis. Mol. Biol. Cell, Dec 2007; 18: 4847 - 4858. |
HeLa | Cervical Carcinoma | Human | GeneSilencer siRNA Transfection Reagent | Cassimeris, L. and Morabito, J. (2004) TOGp, the Human Homolog of XMAP215/Dis1, Is Required for Centrosome Integrity, Spindle Pole Organization, and Bipolar Spindle Assembly. Mol. Biol. Cell 15 (4): 1580-1590. |
HeLa | Cervical Carcinoma | Human | GeneSilencer siRNA Transfection Reagent | Chung, J-S., Sato, K., Dougherty, I.I., Cruz, Jr., P.D. and Ariizumi, K. (2007) DC-HIL is a negative regulator of T lymphocyte activation. Blood. 109(10): p. 4320-4327. |
HeLa | Cervical Carcinoma | Human | GeneSilencer siRNA Transfection Reagent | Kheifets, V., Bright, R., Inagaki, K., Schechtman, D. and Mochly-Rosen, D. (2006) Protein Kinase C δ}PKC)-Annexin V Interaction: a required step in δ PKC translocation and function. J. Biol. Chem. 281(32): p. 23218-23226. |
HeLa | Cervical Carcinoma | Human | GeneSilencer siRNA Transfection Reagent | Layzer, J.M., McCaffrey, A.P., Tanner, A.K., Huang, Zan, Kay, Mark A. and Sullenger, B.A. (2004) In vivo activity of nuclease-resistant siRNAs. RNA 10 (5): p. 766-771. |
HeLa | Cervical Carcinoma | Human | GeneSilencer siRNA Transfection Reagent | Pomerening, J.R., Ubersax, J.A. and Ferrell, J.E., Jr. (2008) Rapid Cycling and Precocious Termination of G1 Phase in Cells Expressing CDK1AF. Mol. Biol. Cell, 19: 3426 - 3441. |
HeLa | Cervical Carcinoma | Human | GeneSilencer siRNA Transfection Reagent | Shulga, N. and Pastorino, J.G. (2006) Acyl Coenzyme A-binding Protein Augments Bid-induced Mitochondrial Damage and Cell Death by Activating μ-Calpain. J. Biol. Chem. 281(41): 30824-30833. |
HeLa | Cervical Carcinoma | Human | GeneSilencer siRNA Transfection Reagent | Witherow, D.S., Garrison, T.R., Miller, W.E. and Robert J. Lefkowitz (2004) {beta}-Arrestin inhibits NF-{kappa}B activity by means of its interaction with the NF-{kappa}B inhibitor I{kappa}B{alpha}. Proc. Natl. Acad. Sci. USA 101: 8603-8607. |
HeLa | Cervical Carcinoma | Human | GeneSilencer siRNA Transfection Reagent | Yang, Q-H, Church-Hajduk, R., Ren, J., Newton, M.L. and Du, C. (2003) Omi/HtrA2 catalytic cleavage of inhibitor of apoptosis (IAP) irreversibly inactivates IAPs and facilitates caspase activity in apoptosis. Genes & Dev. 17: 1487 - 1496. |
HeLa | Cervical Carcinoma | Human | GeneSilencer siRNA Transfection Reagent | Zhu, S., Wang, W., Clarke, D.C. and Liu, X. (2007) Activation of Mps1 Promotes Transforming Growth Factor-β-independent Smad Signaling. J. Biol Chem. 282 (25) 18327-18338. |
HepG2 | Hepatocellular Carcinoma | Human | GeneSilencer siRNA Transfection Reagent | Pastorino, J.G. and Shulga, N. (2008) TNFalpha can provoke cleavage and activation of sterol regulatory element binding protein in ethanol exposed cells via a caspase dependent pathway that is cholesterol-insensitive. J. Biol. Chem. 283: 25638 - 25649. |
HL-60 | Peripheral Blood Promyelocytic Leukemia | Human | GeneSilencer siRNA Transfection Reagent | Smirnova, I.V., Kajstura, M., Sawamura, T. and Goligorsky, M.S. (2004) Asymmetric dimethylarginine upregulates LOX-1 in activated macrophages: role in foam cell formation. AJP: Heart 287(2): p. H782-H790 |
HMEC-1 | Human Microvascular Endothelial Cells | Human | GeneSilencer siRNA Transfection Reagent | Zhou, X., Stuart, A., Dettin, L.E., Rodriguez, G., Hoel, B. and Gallicano G.I. (2004) Desmoplakin is required for microvascular tube formation in culture. J. Cell Sci. 117: 3129-3140. |
HPAEC | Pulmonary Artery Endothelium | Human | GeneSilencer siRNA Transfection Reagent | Berdyshev, E.V., Gorshkova, I., Skobeleva, A., Bittman, R., Lu, X., Dudek, S.M., Mirzapoiazova, T., Garcia, J.G.N. and Natarajan, V. (2009) FTY720 inhibits ceramide synthases and upregulates dihydrosphingosine-1-phosphate formation in human lung endothelial cells. J. Biol. Chem. 284 (9): 5467-5477. |
HT29 | Colon Adenocarcinoma | Human | GeneSilencer siRNA Transfection Reagent | Yao, K., Shida, S., Selvakumaran, M., Zimmerman, R., Simon, E., Schick, J., Haas, N.B. Balke, M., Ross, H., Johnson, S.W. and O"Dwyer, P.J. (2005) Macrophage Migration Inhibitory Factor Is a Determinant of Hypoxia-Induced Apoptosis in Colon Cancer Cell Lines. Clin. Can. Res. 11: 7264-7272. |
HUT-78 | T Cells | Human | GeneSilencer siRNA Transfection Reagent | Maneechotesuwan, K., Xin, Y., Ito, K., Jazrawi, E., Lee, K-Y., Usmani, O.S., Barnes, P.J. and Adcock, I.M. (2007) Regulation of Th2 Cytokine Genes by p38 MAPK-Mediated Phosphorylation of GATA-3. J. Immunol. 178(4): 2491-2498. |
JAR | Choriocarcinoma | Human | GeneSilencer siRNA Transfection Reagent | Abboud-Jarrous, G., Atzmon, R., Peretz, T., Palermo, C., Gadea, B.B., Joyce, J.A. and Vlodavsky, I. (2008) Cathepsin L Is Responsible for Processing and Activation of Proheparanase through Multiple Cleavages of a Linker Segment. J. Biol. Chem. 283: 18167 - 18176. |
Jurkat | T-Cell Lymphoma | Human | GeneSilencer siRNA Transfection Reagent | Nguyen, J.T. and Wells, J.A. (2003) Direct activation of the apoptosis machinery as a mechanism to target cancer cells. Proc. Natl. Acad. Sci. 100: 7533 7538. |
KATOIII | Gastric Epithelium | Human | GeneSilencer siRNA Transfection Reagent | Nagasako, T., Sugiyama, T., Mizushima, T., Miura, Y., Kato, M. and Asaka, M. (2003) Up-regulated Smad5 Mediates Apoptosis of Gastric Epithelial Cells Induced by Helicobacter pylori Infection. J. Biol. Chem: 278: 4821 - 4825. |
L/Stab-2 | Fibroblast | Mouse | GeneSilencer siRNA Transfection Reagent | Park, S-Y., Kang, K-B., Thapa, N., Kim, S-Y., Lee, S-J. and Kim, I-S. (2008) Requirement of Adaptor Protein GULP during Stabilin-2-mediated Cell Corpse Engulfment. J. Biol. Chem. 283(16): 10593-10600. |
LNCaP | Prostate Cancer | Human | GeneSilencer siRNA Transfection Reagent | Takayama, K., Tsutsumi, S., Suzuki, T., Horie-Inoue, K., Ikeda, K., Kaneshiro, K. Fujimura, T., Kumagai, J., Urano, T., Sakaki, Y., Shirahige, K., Sasano, H., Takahashi, S., Kitamura, T., Ouchi, Y., Aburatani, H. and Inoue, S. (2009) Amyloid precursor protein is a primary androgen target gene that promotes prostate cancer growth. Cancer Res 1 69(1): p. 137. |
M2 | Melanoma | Human | GeneSilencer siRNA Transfection Reagent | Scott, M.G.H., Pierotti, V., Storez, H., Lindberg, E., Thuret, A., Muntaner, O., Labbe-Jullie, C., Pitcher, J.A. and Marullo, S. (2006) Cooperative Regulation of Extracellular Signal-Regulated Kinase Activation and Cell Shape Change by Filamin A and β-Arrestins. Mol. & Cell Biol, 26: 3432-3445. |
McA-RH 7777 | Hepatoma | Rat | GeneSilencer siRNA Transfection Reagent | Pastorino, J.G. and Shulga, N. (2008) Tumor Necrosis Factor-{alpha} Can Provoke Cleavage and Activation of Sterol Regulatory Element-binding Protein in Ethanol-exposed Cells via a Caspase-dependent Pathway That Is Cholesterol Insensitive. J. Biol. Chem. 283: 25638 - 25649. |
MCF-7 | Breast Adenocarcinoma | Human | GeneSilencer siRNA Transfection Reagent | Ikeda, K., Ogawa, S., Tsukui, T., Horie-Inoue, K., Ouchi, Y., Kato, S., Muramatsu, M. and Inoue, S. (2004) Protein Phosphatase 5 Is a Negative Regulator of Estrogen Receptor-mediated Transcription. Mol. Endocrinol. 18(5): 1131-1143. |
MDA MB-231 | Breast Cancer | Human | GeneSilencer siRNA Transfection Reagent | Ge, L., Shenoy, S.K., Lefkowitz, R.J. and DeFea, K.A. (2004) Constitutive protease-activated-receptor-2 mediated migration of MDA MB-231 breast cancer cells requires both beta-arrestin-1 and 2. J. Biol. Chem. 279(53): p. 55419-55424. |
MDA MB-468 | Breast Carcinoma | Human | GeneSilencer siRNA Transfection Reagent | Ge, L., Shenoy, S.K., Lefkowitz, R.J. and DeFea, K.A. (2004) Constitutive protease-activated-receptor-2 mediated migration of MDA MB-231 breast cancer cells requires both beta-arrestin-1 and 2. J. Biol. Chem.279(53): p. 55419-55424. |
MDA-MB-435 | Melanoma | Human | GeneSilencer siRNA Transfection Reagent | Abboud-Jarrous, G., Atzmon, R., Peretz, T., Palermo, C., Gadea, B.B., Joyce, J.A.and Vlodavsky, I. (2008) Cathepsin L Is Responsible for Processing and Activation of Proheparanase through Multiple Cleavages of a Linker Segment. J. Biol. Chem. 283: 18167 - 18176. |
MDA-MB-468 | Breast Carcinoma | Human | GeneSilencer siRNA Transfection Reagent | Zoudilova, M., Kumar, P., Ge, L., Wang, P., Bokoch, G. M. and DeFea, K. A. (2007) β-arrestin-dependent regulation of the cofilin pathway downstream of protease-activated receptor-2. J. Biol. Chem. 282: 20634 - 20646. |
MG63 | Osteosarcoma | Human | GeneSilencer siRNA Transfection Reagent | Ichikawa, T., Horie-Inoue, K., Ikeda, K., Blumberg, B. and Inoue, S. (2007) Vitamin K2 induces phosphorylation of protein kinase A and expression of novel target genes in osteoblastic cells. J Mol Endocrinol 1 39 (4): 239. |
MKN 28 | Gastric Epithelium | Human | GeneSilencer siRNA Transfection Reagent | Nagasako, T., Sugiyama, T., Mizushima, T., Miura, Y., Kato, M. and Asaka, M. (2003) Up-regulated Smad5 Mediates Apoptosis of Gastric Epithelial Cells Induced by Helicobacter pylori Infection. J. Biol. Chem.: 278: 4821 - 4825. |
MKN 45 | Gastric Epithelium | Human | GeneSilencer siRNA Transfection Reagent | Nagasako, T., Sugiyama, T., Mizushima, T., Miura, Y., Kato, M. and Asaka, M. (2003) Up-regulated Smad5 Mediates Apoptosis of Gastric Epithelial Cells Induced by Helicobacter pylori Infection. J. Biol. Chem.: 278: 4821 - 4825. |
MV-4-11 | Biophenotypic B Myelomonocytic Leukemia | Human | GeneSilencer siRNA Transfection Reagent | Yang, X., Liu, L., Sternberg, D., Tang, L., Galinsky, I., DeAngelo, D. and Stone, R. (2005) The FLT3 Internal Tandem Duplication Mutation Prevents Apoptosis in Interleukin-3-Deprived BaF3 Cells Due to Protein Kinase A and Ribosomal S6 Kinase 1-Mediated BAD Phosphorylation at Serine 112. Cancer Res. 65(16): p. 7338-7347. |
Neuro2a | Neuroblastoma | Mouse | GeneSilencer siRNA Transfection Reagent | Bertling, E., Hotulainen, P., Mattila, P.K., Matilainen, T., Salminen, M., and Lappalainen, P. (2004) Cyclase-associated-protein 1 (CAP1) promotes cofilin-induced actin dynamics in mammalian nonmuscle cells. Mol. Biol. Cell 15 (5): p. 2324-2334. |
NIH-3T3 | Fibroblast | Mouse | GeneSilencer siRNA Transfection Reagent | Bertling, E., Hotulainen, P., Mattila, P.K., Matilainen, T., Salminen, M., and Lappalainen, P. (2004) Cyclase-associated-protein 1 (CAP1) promotes cofilin-induced actin dynamics in mammalian nonmuscle cells. Mol. Biol. Cell 15 (5): 2324-2334. |
NIH-3T3 | Fibroblast | Mouse | GeneSilencer siRNA Transfection Reagent | Hotulainen, P., Paunola, E., Vartiainen, M.K. and Lappalainen, P. (2005) Actin-depolymerizing Factor and Cofilin-1 Play Overlapping Roles in Promoting Rapid F-Actin Depolymerization in Mammalian Nonmuscle Cells. Mol. Biol. Cell. 16(2): p. 649-664. |
NIH-3T3 | Fibroblast | Mouse | GeneSilencer siRNA Transfection Reagent | Nomachi, A., Nishita, M., Inaba, D., Enomoto, M., Hamasaki, M. and Minami, Y. (2008) Receptor Tyrosine Kinase Ror2 Mediates Wnt5a-induced Polarized Cell Migration by Activating c-Jun N-terminal Kinase via Actin-binding Protein Filamin A. J. Biol. Chem. 283(41): 27973-27981. |
OK | Kidney | Opossum | GeneSilencer siRNA Transfection Reagent | Khundmiri, S.J., Ameen, M., Delamere, N.A. and Lederer, E.D. (2008) PTH-mediated regulation of Na+ -K+ -ATPase requires Src kinase-dependent ERK Phosphorylation. Am J Physiol Renal Physiol. 295(2): F426-F437. |
OK | Kidney | Opossum | GeneSilencer siRNA Transfection Reagent | Khundmiri, S.J., Dean, W.L., McLeish, K.R. and Lederer, E.D. (2005) Parathyroid Hormone-mediated Regulation of Na+-K+-ATPase Requires ERK-dependent Translocation of Protein Kinase C{alpha}. J. Biol. Chem. 280(10): p. 8705-8713. |
PC-12 | Pheochromocytoma | Rat | GeneSilencer siRNA Transfection Reagent | de Barry, J., Janoshazi, A., Luc Dupont, J., Procksch, O., Chasserot-Golaz, S., Jeromin, A. and Vitale, N. (2006) Functional Implication of Neuronal Calcium Sensor-1 and Phosphoinositol 4-Kinase-β Interaction in Regulated Exocytosis of PC12 Cells. J. Biol Chem. 281(26): 18098-18111. |
Primary | Cortical Neurons | Mouse | GeneSilencer siRNA Transfection Reagent | Aarts, M., Lihara, K., Wei, W-L, Xiong, Z-G, Arundine, M., Cerwinski, W., MacDonald, J.F., Tymianski, M. (2003) A Key Role for TRPM7 Channels in Anoxic Neuronal Death. Cell 115: 863. |
Primary | Cortical Neurons | Mouse | GeneSilencer siRNA Transfection Reagent | Aleyasin, H., Cregan, S.P., Iyirhiaro, G., OHare, M.J., Callaghan, S.M., Slack, R.S. and Park, D.S. (2004) Nuclear Factor-B Modulates the p53 Response in Neurons Exposed to DNA Damage. J. Neurosci. 24 (12): 2963-2973. |
Primary | Hippocampal Neurons | Rat | GeneSilencer siRNA Transfection Reagent | Amaral, M.D. and Pozzo-Miller, L. (2007) TRPC3 Channels Are Necessary for Brain-Derived Neurotrophic Factor to Activate a Nonselective Cationic Current and to Induce Dendritic Spine Formation. J. Neurosci. 27(19): p. 5179-5189. |
Primary | Macrophage | Human | GeneSilencer siRNA Transfection Reagent | Asmis, R., Wang, Y., Xu, L., Kisgati, M., Begley, J.G. and驴Mieyal, J.J. (2005) A novel thiol oxidation-based mechanism for adriamycin-induced cell injury in human macrophages. FASEB J. published 13 September 2005, 10.1096/fj.04-2991fje. |
Primary | Pulmonary Artery Endothelium | Human | GeneSilencer siRNA Transfection Reagent | Birukova, A.A., Chatchavalvanich, S., Rios, A., Kawkitinarong, K., Garcia, J.G.N. and Birukov, K.G. (2006) Differential Regulation of Pulmonary Endothelial Monolayer Integrity by Varying Degrees of Cyclic Stretch. Am. J. Pathol. 168: 1749 - 1761. |
Primary | Cortical Neurons | Mouse | GeneSilencer siRNA Transfection Reagent | Burkhalter, J., Fiumelli, H., Erickson, J.D. and Martin, J-L. (2007) A critical role for system a amino acid transport in the regulation of dendritic development by BDNF. J Biol Chem 282: 5152 - 5159. |
Primary | Dendritic Cells (Bone Marrow) | Mouse | GeneSilencer siRNA Transfection Reagent | Chung, J-S, Sato, K., Dougherty, I.I., Cruz, Jr., P.D. and Ariizumi, K. (2007) DC-HIL is a negative regulator of T lymphocyte activation. Blood. 109(10): p. 4320-4327. |
Primary | Cortical Neurons | Mouse | GeneSilencer siRNA Transfection Reagent | Cui, H., Hayashi, A., Sun, H-S, Belmares, M.P., Cobey, C., Phan, T., Schweizer, J., Salter, M.W., Wang, Y.T., Tasker, R.A., Garman, D., Rabinowitz, J., Lu, P.S. and Tymianski, M. (2007) PDZ Protein Interactions Underlying NMDA Receptor-Mediated Excitotoxicity and Neuroprotection by PSD-95 Inhibitors. J. Neurosci. 27(37): 9901-9915. |
Primary | Coronary Artery Endothelial | Human HCAEC | GeneSilencer siRNA Transfection Reagent | Dandapat, A., Hu, C., Sun, L. and Mehta, J.L. (2007) Small Concentrations of oxLDL Induce Capillary Tube Formation From Endothelial Cells via LOX-1 Dependent Redox-Sensitive Pathway. Arterioscler Thromb Vasc Biol. |
Primary | Peritoneal Macrophage | Mouse | GeneSilencer siRNA Transfection Reagent | de Beer, M.C., Zhao, Z., Webb, N.R., van der Westhuyzen, D.R. and de Villiers, W.J.S. (2003) Lack of a direct role for macrosialin in oxidized LDL metabolism J. Lipid Res. 44: 674 - 685. |
Primary | Dorsal Root Ganglion Neurons | Mouse | GeneSilencer siRNA Transfection Reagent | Haruhisa Higuchi, Toshihide Yamashita, Hideki Yoshikawa, and Masaya Tohyama (2003) Functional inhibition of the p75 receptor using a small interfering RNA. Biochem. & Biophys. Res. Comm. 301: 804809. |
Primary | Retinal Ganglion | Rat | GeneSilencer siRNA Transfection Reagent | Hayashi, H., Campenot, R.B., Vance, D.E. and Vance, J.E. (2007) Apolipoprotein E-Containing Lipoproteins Protect Neurons from Apoptosis via a Signaling Pathway Involving Low-Density Lipoprotein Receptor-Related Protein-1. J. Neurosci. 27(8): 1933-1941. |
Primary | Dorsal Root Ganglion | Rat | GeneSilencer siRNA Transfection Reagent | Hengst, U. Cox, L.J., Macosko, E.Z. and Jaffrey, S.R. (2006) Functional and Selective RNA Interference in Developing Axons and Growth Cones. J. Neurosci. 26(21): 5727-5732. |
Primary | Schwann Cells | Mouse | GeneSilencer siRNA Transfection Reagent | Higuchi, H., Yamashita, T., Yoshikawa, H. and Tohyama, M. (2003) Functional inhibition of the p75 receptor using a small interfering RNA. Biochem. & Biophys. Res. Comm. 301: 804809. |
Primary | Cardiac Fibroblast | Mouse | GeneSilencer siRNA Transfection Reagent | Hu, C., Dandapat, A., Sun, L., Khan, J.A., Liu, Y., Hermonat, P.L. and Mehta, J.L. (2008) Regulation of TGFbeta 1-mediated collagen formation by LOX-1: Studies based on forced over-expression of TGFbeta 1 in wild-type and LOX-1 knockout mice cardiac fibroblasts. J. Biol. Chem. 283 (16) 10226-10231. |
Primary | Cardiac Myocytes (Neonatal) | Rat | GeneSilencer siRNA Transfection Reagent | Juhaszova, M., Zorov, D.B., Kim, S-H, Pepe, S., Fu, O., Fishbein, K.W., Ziman, B.D., Wang, S., Ytrehus, K., Antos, C.L., Olson, E.N. and Sollott, S.J. (2004) Glycogen synthase kinase-3{beta} mediates convergence of protection signaling to inhibit the mitochondrial permeability transition pore. J. Clin. Invest. 113(11): p. 1535-1549. |
Primary | Lung Microvascular Endothelial Cells | Human | GeneSilencer siRNA Transfection Reagent | Kolosova, I.A., Ma, S-F, Adyshev, D.M., Wang, P., Ohba, M., Natarajan, V., Garcia, J.G.N. and Verin, A.D. (2004) Role of CPI-17 in the regulation of endothelial cytoskeleton AJP: Lung. 287(5): L970. |
Primary | Aortic Muscle Cells | Rat | GeneSilencer siRNA Transfection Reagent | Lake, A.C. & Castellot, J.J. (2003) CCN5 modulates the antiproliferative effect of heparin and regulates cell motility in vascular smooth muscle cells. Cell Comm. & Signaling 1: 5. |
Primary | Dendritic Cells | Mouse | GeneSilencer siRNA Transfection Reagent | Li, M., Qian, H., Ichim, T.E., Ge, W-W, Popov, I.A., Rycerz, K., Neu, J., White, D., Zhong, R., Min, W-P. (2004) Induction of RNA Interference in Dendritic Cells. Immunologic Research 30 (2) 215-230. |
Primary | Dendritic Cells | Mouse | GeneSilencer siRNA Transfection Reagent | Li, M., Zhang, X., Zheng, X., Lian, D., Zhang, Z-X., Ge, W., Yang, J., Vladau, C., Suzuki, M., Chen, D., Zhong, R., Garcia, B., Jevnikar, A.M. and Min, W-P (2007) Immune Modulation and Tolerance Induction by RelB-Silenced Dendritic Cells through RNA Interference. J. Immunol. 178(9): p. 5480-5487. |
Primary | Dendritic Cells | Mouse | GeneSilencer siRNA Transfection Reagent | Liu, G., Ng, H., Akasaki, Y., Yuan, X., Ehtesham, M., Yin, D., Black , K.L. and Yu, J.S. (2004) Small interference RNA modulation of IL-10 in human monocyte-derived dendritic cells enhances the Th1 response. Eur. J. Immunol. 34: 1680-1687. |
Primary | Cerebellar Neurons | Rat | GeneSilencer siRNA Transfection Reagent | Numakawa, T., Nakayama, H., Suzuki, S., Kubo, T., Nara, F., Numakawa, Y., Yokomaku, D., Araki, T., Ishimoto, T., Ogura, A. and Taguchi, T. (2003) Nerve growth factor-induced glutamate release is via p75 receptor, ceramide and Ca2+ from ryanodine receptor in developing cerebellar neurons. J. Biol. Chem: 278: 41259-41269. |
Primary | Osteoblast | Mouse | GeneSilencer siRNA Transfection Reagent | Ohyama, Y., Nifuji, A., Maeda, Y., Amagasa, T. and Noda, M. (2004) Spaciotemporal Association and Bone Morphogenetic Protein Regulation of Sclerostin and Osterix Expression during Embryonic Osteogenesis. Endocrinology. 145(10): p. 4685-4692. |
Primary | HMVEC | Human | GeneSilencer siRNA Transfection Reagent | Radu Stefanescu, Dustin Bassett, Rozbeh Modarresi, Francisco Santiago, Mohamad Fakruddin, and Jeffrey Laurence (2008) Synergistic interactions between interferon-{gamma} and TRAIL modulate c-FLIP in endothelial cells, mediating their lineage-specific sensitivity to thrombotic thrombocytopenic purpura plasma-associated apoptosis. Blood 112: 340 - 349. |
Primary | Cardiac Myocytes | Feline | GeneSilencer siRNA Transfection Reagent | Ramabadran, R. S., Chancey, A., Vallejo, J.G., Barger, P.M., Sivasubramanian, N. and Mann, D.L. (2008) Targeted Gene Silencing of Tumor Necrosis Factor Attenuates the Negative Inotropic Effects of Lipopolysaccharide in Isolated Contracting Cardiac Myocytes. Tex Heart Inst J. 35(1): 16-21. |
Primary | T-Cells (CD3+) | Human | GeneSilencer siRNA Transfection Reagent | Samten, B., Howard, S.T., Weis, S.E., Wu, S., Shams, H., Townsend, J.C., Safi, H. and Barnes, P.F. (2005) Cyclic AMP Response Element-Binding Protein Positively Regulates Production of IFN-{gamma} by T Cells in Response to a Microbial Pathogen. J. Immunol. 174(10): p. 6357-6363. |
Primary | Cortical Neurons | Mouse | GeneSilencer siRNA Transfection Reagent | Sato, S., Xu, J., Okuyama, S., Martinez, LB., Walsh, S.M, Jacobsen, M.T, Swan, R.J., Schlautman, J.D., Ciborowski, P. and Ikezu, T. (2008) Spatial learning impairment, enhanced CDK5/p35 activity, and downregulation of NMDA receptor expression in transgenic mice expressing tau-tubulin kinase 1. J Neurosci 28(53): 14511. |
Primary | Dendritic Cells | Human | GeneSilencer siRNA Transfection Reagent | Smith, A.L., Ganesh, L., Leung, K., Jongstra-Bilen, J., Jongstra, J. and Nabel, G.J. (2007) Leukocyte-specific protein 1 interacts with DC-SIGN and mediates transport of HIV to the proteasome in dendritic cells. J. Exp. Med. 204(2): p. 421-430. |
Primary | Cortical Neurons | Mouse | GeneSilencer siRNA Transfection Reagent | Supnet, C., Grant, J., Kong, H., Westaway, D. and Mayne, M. (2006) Amyloid β-(1-42) Increases Ryanodine Receptor-3 Expression and Function in TgCRND8 Mice. J. Biol. Chem. 281: 38440 - 38447. |
Primary | Aveolar Type II Cells | Mouse | GeneSilencer siRNA Transfection Reagent | Ueno, T., Linder, S., Na, C-L, Rice, W.R., Johansson, J., and Weaver, T.E. (2004) Processing of Pulmonary Surfactant Protein B by Napsin and Cathepsin H. J. Biol. Chem. 279: 16178-16184. |
Primary | Macrophage | Mouse | GeneSilencer siRNA Transfection Reagent | Wang, Y., Chen, T., Han, C., He, D., Liu, H., An, H., Cai, Z. and Cao, X. (2007) Lysosome-associated small Rab GTPase Rab7b negatively regulates TLR4 signaling in macrophages by promoting lysosomal degradation of TLR4. Blood 110: 962 - 971. |
Primary | Dorsal Root Ganglion | Rat | GeneSilencer siRNA Transfection Reagent | Wu, K.Y., Hengst, U., Cox, L.J., Macosko, E.Z., Jeromin, A., Urquhart, E.R., and Jaffrey, S.R. (2005) Local translation of RhoA regulates growth cone collapse. Nature 436: 1020-1024. |
Primary | Lacrimal Gland Acinar Cells | Rabbit | GeneSilencer siRNA Transfection Reagent | Xie, J., Chiang, L., Contreras, J., Wu, K., Garner, J.A., Medina-Kauwe, L. and Hamm-Alvarez, S.F. (2006) Novel Fiber-Dependent Entry Mechanism for Adenovirus Serotype 5 in Lacrimal Acini. J. Virol. 80(23): 11833 - 11851. |
Primary | Bone Marrow | Mouse | GeneSilencer siRNA Transfection Reagent | Yang, R., Cai, Z., Zhang, Y., Yutzy IV, W.H., Roby, K.F. and Roden, R.B.S. (2006) CD80 in Immune Suppression by Mouse Ovarian Carcinoma驴Associated Gr-1+CD11b+ Myeloid Cells. Cancer Res 66 (13): 6807 - 6815. |
Primary | Dendritic Cells | Mouse | GeneSilencer siRNA Transfection Reagent | Zheng, X., Koropatnick, J., Li, M., Zhang, X., Ling, F., Ren, X., Hao, X., Sun, H., Vladau, C., Franek, J.A., Feng, B., Urquhart, B.L., Zhong, R., Freeman, D.J., Garcia, B. and Min, W-P. (2006) Reinstalling Antitumor Immunity by Inhibiting Tumor-Derived Immunosuppressive Molecule IDO through RNA Interference. J. Immunol. 177(8): 5639-5646. |
Primary | Pulmonary Artery Endothelium | Human | GeneSilencer siRNA Transfection Reagent | Zhuowei Li, Xhevahire Hyseni, Jacqueline D. Carter, Joleen M. Soukup, Lisa A. Dailey, and Yuh-Chin T. Huang (2006) Pollutant particles enhanced H2O2 production from NAD(P)H oxidase and mitochondria in human pulmonary artery endothelial cells. Am J Physiol Cell Physiol 291: C357 - C365. |
Primary HPAEC | Pulmonary Artery Endothelium | Human | GeneSilencer siRNA Transfection Reagent | Rentsendorj, O., Mirzapoiazova, T., Adyshev, D., Servinsky, L.E., Renne, T., Verin, A.D. and Pearse, D.B. (2008) Role of vasodilator-stimulated phosphoprotein in cGMP-mediated protection of human pulmonary artery endothelial barrier function. Am J Physiol Lung Cell Mol Physiol. 294(4): L686 -L697. |
RAW 264.7 | Macrophage | Mouse | GeneSilencer siRNA Transfection Reagent | de Beer, M.C., Zhao, Z., Webb, N.R., van der Westhuyzen, D.R. and de Villiers, W.J.S. (2003) Lack of a direct role for macrosialin in oxidized LDL metabolism. J. Lipid Res. 44: 674 - 685. |
RAW 264.7 | Macrophage | Mouse | GeneSilencer siRNA Transfection Reagent | Liu, X., Yao, M., Li, N., Wang, C., Zheng, Y. and Cao, X. (2008) CaMKII promotes TLR-triggered proinflammatory cytokine and type I interferon production by directly binding and activating TAK1 and IRF3 in macrophages. Blood. 112(13): 4961-4970. |
Renca | Renal Cell Carcinoma | Mouse | GeneSilencer siRNA Transfection Reagent | Ogushi, T., Takahashi, S., Takeuchi, T., Urano, T., Horie-Inoue, K., Kumagai, J., Kitamura, T., Ouchi, Y., Muramatsu, M. and Inoue, S. (2005) Estrogen Receptor-Binding Fragment-Associated Antigen 9 Is a Tumor-Promoting and Prognostic Factor for Renal Cell Carcinoma. Cancer Res. 65(9): p. 3700-3706. |
SaOs2 | Osteosarcoma | Human | GeneSilencer siRNA Transfection Reagent | Chu, F.,Chou, P.M., Zheng, X., Mirkin, B.L. and Rebbaa, A.驴(2005)驴Control of Multidrug Resistance Gene mdr1 and Cancer Resistance to Chemotherapy by the Longevity Gene sirt1. Cancer Res.65 (22): p. 10183-10187. |
SaOS2 | Osteosarcoma | Human | GeneSilencer siRNA Transfection Reagent | Zheng, X., Chu, F., Chou, P.M., Gallati, C., Dier, U., Mirkin, B.L., Mousa, S.A. and Rebbaa, A. (2009) Cathepsin L inhibition suppresses drug resistance in vitro and in vivo: a putative mechanism. Am J Physiol Cell Physiol. 296(1): C65-C74. |
Saos-2 | Osteosarcoma | Human | GeneSilencer siRNA Transfection Reagent | Zheng, X., Chou, P.M., Mirkin, B.L. and Rebbaa, A. (2004) Senescence-initiated Reversal of Drug Resistance: Specific Role of Cathepsin L. Cancer Res. 64: 1773 - 1780. |
SCCH196 | Sarcoma | Human | GeneSilencer siRNA Transfection Reagent | Takahashi, A., Higashino, F., Aoyagi, M., Yoshida, K., Itoh, M., Kyo, S., Ohno, T., Taira, T., Ariga, H., Nakajima, K., Hatta, M., Kobayashi, M., Sano, H., Kohgo, T. and Shindoh, M., (2003) EWS/ETS Fusions Activate Telomerase in Ewings Tumors. Cancer Res. 63: 8338-8344. |
SNB19 | Glioblastoma | Human | GeneSilencer siRNA Transfection Reagent | Song, S.W., Fuller, G.N., Zheng, H. and Zhang, W. (2005) Inactivation of the Invasion Inhibitory Gene IIp45 by Alternative Splicing in Gliomas. Cancer Res. 65(9): p. 3562-3567. |
SW480 | Colon Adenocarcinoma | Human | GeneSilencer siRNA Transfection Reagent | Zhu, S., Wang, W., Clarke, D.C. and Liu, X. (2007) Activation of Mps1 Promotes Transforming Growth Factor-β-independent Smad Signaling. J. Biol. Chem. 282(25): 18327-18338. |
U20S | Osteosarcoma | Human | GeneSilencer siRNA Transfection Reagent | Hotulainen P. and Lappalainen, P. (2006) Stress fibers are generated by two distinct actin assembly mechanisms in motile cells J. Cell Biol. 173(3): 383-394. |
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第一代热启动Taq酶是一种DNA聚合酶。 第一代指的是直接从Taq(这是一种细菌)体内提取的天然聚合酶,没有进行化学修饰的,为了达到更好的聚合催化效果,往往需要经过酶工程处理,得到的聚合酶催化效率更高。 热启动是指这种酶的反应温度条件
Taq DNA polymerase是从克隆有Thermu aquaticus DNA Polymerase基因的大肠杆菌经诱导表达后,再经过柱纯化分离出来的一个约94 KD的重组蛋白。无外源核酸酶和细菌DNA污染,稳定性好,特异性强,适用于常规PCR扩增。
摘要:随着转基因技术的发展,获取目的基因片段的方法也越来越多样化,获取目的基因片段的技术也日新月异,但是无论获取的方法和手段怎么样发展,获取的主要思路始终在围绕一根主线在开展。
关键词:基因 ,遗传效应,基因克隆,体外重组
一、基因工程
基因工程作为一门理论性与实践性较强的学科,其方法与技术已经渗透到现代生命科学的各个分支领域,成为生命科学的一门核心技术。基因工程包含许多独特的实验方法和技术,不仅内容丰富,涉及面广,实用性也强。基因工程是通过DNA 重组技术, 获得具有特殊生物遗传性状和功能的遗传工具生物体, 基因工程技术广泛应用于农业、医学、食品工业等。本文就基因工程的应用现状综合阐述。
基因工程技术是一项极为复杂的高新生物技术, 它利用现代遗传学与分子生物学的理论和方法, 按照人类所需, 用DNA 重组技术对生物基因组的结构和组成进行人为修饰或改造, 从而改变生物的结构和功能, 使之有效表达出人类所需要的蛋白质或人类有益的生物性状。基因工程从诞生至今, 仅有30 年的历史, 然而, 无论是在基础理论研究领域, 还是在生产实际应用方面, 都已取得了惊人的成绩。首先,基因工程给生命科学自身的研究带来了深刻的变化。目前科学家已完成了多种细胞器的基因组全序列测定工作。其次, 基因工程具有广泛的应用价值, 能为工农业生产、医药卫生、环境保护开辟新途径。
基因工程( 又称DNA 重组技术、基因重组技术) , 是20 世纪70 年代初兴起的技术科学, 是用人工的方法将目的基因与载体进行DNA重组, 将DNA 重组体送入受体细胞, 使它在受体细胞内复制、转录、翻译, 获得目的基因的表达产物。这种跨越天然物种屏障, 把来自任何生物的基因置于毫无亲缘关系的新的寄主生物细胞之中的能力, 是基因工程技术区别于其他技术的根本特征。 基因工程研究内容为:
(1) 从复杂的生物有机体基因组中, 经过酶切消化或PCR 扩增等步骤, 分离出带有目的基因的DNA 片段。
(2) 在体外, 将带有目的基因的外源DNA 片段连接到能够自我复制并具有选择记号的载体分子上, 形成重组DNA分子。
(3)重组DNA 分子转移到适当的受体细胞, 并与之一起增殖。
(4) 从大量的细胞繁殖群体中, 筛选出获得了重组DNA 分子的受体细胞克隆。
目前常用方法有:化学修饰法、反应动力学法、X-射线衍射法等.1.化学修饰法:此方法是研究最早,应用最广泛的方法.原则上讲,酶分子侧链上的各种基团
限制性核酸内切酶的研究和应用发展很快,已提纯的限制性核酸内切酶有100多种,许多已成为基因工程研究中必不可少的工具酶。
限制性核酸内切酶可被分成三种类型。Ⅰ型和Ⅲ型限制酶水解DNA需要消耗ATP,全酶中的部分亚基有通过在特殊碱基上补加甲基基团对DNA进行化学修饰的活性。
Ⅰ型和Ⅲ型酶具有限制和修饰两种作用,而特异性弱,切割位点的序列不固定,不已知,不宜用于基因克隆中。
Ⅱ型限制酶水解DNA不需要ATP也不以甲基化或其它方式修饰DNA,能在所识别的特殊核苷酸顺序内或附近切割DNA。因此,被广泛用于DNA分子克隆和序列测定。
(1)玻璃制品、塑料制品和电泳槽 灭菌的一次性使用的塑料制品基本上无RNA酶,可以不经预处理直接用于制备和贮存RNA。实验室用的普通玻璃器皿和塑料制品经常有RNA酶法染,使用前必须于180 ℃干烤8小时或更长时间(玻璃器皿)或用氯仿冲洗(塑料制品)。另一种方法是用0.1 %焦碳酸二乙酯(DEPC)的水溶液浸泡用于制备RNA的烧杯,试管和其他用品。DEPC是RNA酶的强烈抑制剂,但其作用并不是绝对的(Fedorcsak和Ehrenberg,1966)。灌满DEPC的玻璃或塑料器皿在37℃放置2小时,然后用灭菌水淋洗数次, 并于100℃干烤15分钟(Kumar和Lindberg,1972)。在15 lbf/in2(1.034x105Pa)高压蒸氯灭菌15分钟。上述处理可以除去器甲上痕量的DEPC,以防DEPC通过羧甲基化作用对RNA的嘌呤碱基进行修饰。
用于RNA电泳的电泳槽应用去污剂洗干净,再用水冲洗,用乙醇干燥,然后灌满3%的H2O2溶液,于室温放置10分钟,然后用0.1 %DEPC处理过的水彻底冲洗电泳槽。最好能留出一些玻璃器皿、塑料制品和电泳槽作上特殊标记,存放在指定地点,为RNA实验专用。
(2)研究人员造成的污染 RNA酶最主要的潜在污染源是研究人员的手。因此,在准备分离的和分析RNA的材料和溶液时,主有涉及RNA的一切操作过程中,都应戴一次性手套,接触“胖的”玻璃器皿和其他物品以后,手套就可能沾染上RNA酶,因此进行RNA实验时应勤换手套。
(3)污染的溶液 用高压灭菌的水和RNA研究专用的化学试剂配制溶液,用干烤过的药匙称取试剂,将溶液装入无RNA酶的玻璃器皿。可能的话溶液均应用0.1%DEPC于37℃至少处理12小时,然后于100℃加热15分钟或在15lbf/in2(1.034x105Pa)的高压下蒸气灭菌15分钟。注:DEPC可与胺类迅速发生化学反应,因些不能用来处理含有Tris 一类的缓冲液。可存几瓶新的未开封的Tris晶体以制备无RNA酶的溶液。
(二)已经污染,可以加入RNA酶的抑制剂
(1)RNA酶的蛋白质抑制剂是 从人胎盘分离的一种蛋白质可与多种RNA酶紧密结合(KI≈3x1010)形成非共价结合的等摩尔复合物,使RNA酶失活。此蛋白质体内可能是血管生成素的抑制剂,血管生成素是氨基酸序列和推测的三级结构与胰RNA酶类似的一种血管生成因子, 几个厂家以不同的商品名出售这种抑制剂,该蛋白质应置于含5mmol/L二硫苏糖醇(DTT)的50%甘油中,贮存于-20℃。抑制剂制品冻融数次后或放置在氧化条件下即应弃之不用,因为上述处理会使蛋白质变性从而释放出所结合的RNA酶。因此,在使用变性剂裂解哺乳动物(mammal;mammalian)细胞这一提取RNA的初始步聚中不应使用这种蛋白抑制剂。然而职用更温和的裂解方法时应使用这种抑制剂,并且在后续的所有RNA纯化步骤中均应有此蛋白质存在。由于酚抽提可以除蛋白质抑制剂,故应在纯化过程中补加几次抑制剂。其最大活性的发挥要求巯基试剂,而且它并不干扰反转录或mRNA在无细胞体系中的翻译。
(2)氧钒核糖核苷复合物 这种由氧钒(1V)离子和4种核糖核苷之中的任意一种所形成的复合物,是量种过渡态类似物,它能与多种RNA酶结合并几科能百分之百地抑制RNA酶的活性。这4种氧钒核糖核苷复合物可加入完整细胞中,在RNA提取和纯化的所有过程中,其使用浓度都是10mmol/L。所得到的mRNA可直接在硅卵母细胞中进行翻译, 并能作为某些外酶促反应(如mRNA反转录)的模板。然而氧钒核糖核苷复合物强烈抑制mRNA在无细胞体系中的翻译,因此必须用含0.1 %羟基喹淋的苯酚[ 用0. 01mol/LTris.Cl(pH7.8)平衡]多次抽提以去除之。有几家公司出售氧钒核糖核苷复合物。
(3)Macaloid(硅藻上)Macaloid是一咱粘土,很多年前就发现它能吸附RNA酶,用缓冲液将其制成浆液,以0.015%(W/V)的终浓度溶解细胞。 这种粘土随同它所吸附的RNA酶可在后续的RNA纯化过程中(如酚抽提后)经离心去除。
(三)提取核酸时可以加入盐酸胍或硫氰酸胍溶液。盐酸胍或硫氰酸胍溶液能迅速溶解蛋白质,从而免受RNA酶的干扰。
限制性酶主要分为三种类型:Ⅰ型限制酶为复合功能酶,具有限制-修饰两种功能,但在 DNA链上没有固定的切割位点,一般在离切割位点1kb到几kb的地方随机切割,不产生特异性片段。Ⅲ型酶与Ⅰ型酶基本相似,不同的是Ⅲ型酶有特异性的切割位点,但这两类酶对 DNA酶切分析的意义不大,通常所说的限制性内切酶是指Ⅱ型酶,它能够识别与切割DNA链上的特定的核苷酸顺序,产生特异性的DNA片段。
2.识别序列及消化产物的末端结构 限制性酶的识别序列,大部分具有双轴对称性结构或称回文序列,如EcoRI的识别序列为:
GAA
TTC
横轴
CTT
AAG
纵轴
将纵轴一侧的序列以横轴为中心旋转180°,则纵轴两侧的序列相互对称,这种结构又称为双重对称结构。大部分酶的识别序列长度为4-6个核苷酸。4核苷酸序列在DNA链中出现频率高,对一随机排列的DNA分子来说,理论值为1/44,因此4核苷酸识别序列的限制性酶在DNA链上切点多,产生片段的数目多,长度短,显示出酶的特异性较低。对于5和6核苷酸识别序列的酶,出现频率分别为1/45和1/46 ,因此,6核苷酸序列在DNA中出现频率低,酶的特异性强,而8核苷酸识别位点在DNA链中出现机率更低(1/48 ),特导性更强,可提供更长的DNA片段。一部分限制性酶具有非典型的双轴对称性序列,其回文识别序列被个或几个其他核苷酸所间隔,如BglⅠ,这种酶的特异性比识别长度相同的典型回文序列的酶略高。另外有些限制性酶(约10种,如BbVⅠ等),其识别序列不表现为回文结构,它们降解双链DNA时,酶切点大部分不在识别序列内,而是与识别序列相距5至13个核苷酸残基不等。
限制性酶切片段的末端结构:限制性酶不但有特定的识别序列,并且任何一种酶切割 DNA链时,总是水解核苷酸3’和5’-磷酸二酯键的3’位磷酸酯键,使产物的5’端带磷酸单酯基团,而3’末端则为游离羟基。因此某一种酶的全部产物的末端具有相同的结构。根据切点序列的结构特点,产物的末端可分为粘性末端和平末端两类。粘性末端指酶切后DNA片段末端带有1-4个核苷酸残基的单链结构,而片段两端突出的单链具有互补性,突出的单链因部位的不同,又可分为5’-与3’-粘性末端两种,突出的单链带5’磷酸单酯的称5’-粘性末端,而突出的单链含3’-羟基则称3’-粘性末端。平末端指酶切后,片段为齐头末端结构。在DNA体外重组时,粘性末端是DNA连接酶的有效底物,有很高的连接效率。向左转|向右转
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