|
| 1. |
Hornakova, T., Staerk, J., Royer, Y., Flex, E., Tartaglia, M., Constantinescu, S.N., Knoops, L. and Renauld, J.C.
(2009)
Acute lymphoblastic leukemia-associated JAK1 mutants activate the Janus kinase/STAT pathway via interleukin-9 receptor alpha homodimers.
J. Biol. Chem.
284
,
6773–6781
.
|
| |
Notes:
The authors studied the ability of JAK1 V658F and A634D mutants to activate the Janus kinase (JAK)/STAT pathway when expressed alone or together with the other components of the interleukin-9 receptor complex. The BOX1 motif of wild-type IL-9Rα, the JAK interacting region, was mutated from PXP to SXS using the GeneEditor™ in vitro Site-Directed Mutagenesis System. To assess STAT transcriptional activity, HEK293 human embryonic kidney, COS-7 monkey kidney, U4C human fibrosarcoma and g2A cells were cotransfected with 250ng of the appropriate constructs, 500ng of firefly luciferase vectors and 50ng of pRL-TK Vector and empty plasmid for a total 1.5µg of DNA. After 24 hours, the cells were lysed in 150µl of Passive Lysis Buffer and reporter activity measured using the Dual-Luciferase® Reporter Assay System. The ProFection® Mammalian Transfection System—Calcium Phosphate was used to transfect 106 HEK293 cells in a six-well plate with 3.75µg of plasmid for Western blot analysis and cotransfected 6 × 106 HEK293 cells in a 100mm dish with 14µg plasmid for immunoprecipitation studies.
(0004025) |
| |
 |
| |
Products: Dual-Luciferase® Reporter 1000 Assay System | Dual-Luciferase® Reporter Assay System | Dual-Luciferase® Reporter Assay System 10-Pack | GeneEditor™ in vitro Site-Directed Mutagenesis System | pRL-TK Vector | ProFection® Mammalian Transfection System—Calcium Phosphate |
| 2. |
Yamada, K., Takahashi, M., Hoshino, Y., Takahashi, H., Ichiyama, K., Tanaka, T. and Okamoto, H.
(2009)
Construction of an infectious cDNA clone of hepatitis E virus strain JE03-1760F that can propagate efficiently in cultured cells.
J. Gen. Virol.
90
,
457–462
.
|
| |
Notes:
The authors created a full-length infectious cDNA clone of a genotype 3 hepatitis E virus (strain JE03-1760F) for use in cell culture. The full-length ORF2 sequence of the JE03-1760F genome was amplified and cloned into the pCI Mammalian Expression Vector. The construct was transfected into PLC/PRF/5 cells for 3 days then analyzed by Western blotting.
(0004029) |
| |
|
| |
Products: pCI Mammalian Expression Vector |
| 3. |
Dall'Osso, C., Guella, I., Duga, S., Locatelli, N., Paraboschi, E.M., Spreafico, M., Afrasiabi, A., Pechlaner, C., Peyvandi, F., Tenchini, M.L. and Asselta, R.
(2008)
Molecular characterization of three novel splicing mutations causing factor V deficiency and analysis of the F5 gene splicing pattern.
Haematologica
93
,
1505–1513
.
|
| |
Notes:
To examine the causes of Factor V (FV) deficiency, the authors examined transcript splicing and its mutated variations. Three regions of human FV (F5) were amplified from a healthy individual and the PCR products cloned into the pTargeT™ Mammalian Expression Vector. Three identified mutations from people with FV deficiency were introduced by site-directed mutatgenesis. All constructs were sequenced before transfection into HeLa cells. After 48 hours, the total RNA was purified and the splicing pattern of the wild type and mutant constructs were analyzed by RT-PCR. The mutant constructs were also transfected into HepG2 cells and tested for nonsense-mediated mRNA decay (NMD) with or without NMD inhibitors (puromycin, cycloheximide, and wortmannin) using RT-PCR.
(0003992) |
| |
|
| |
Products: pTARGET™ Mammalian Expression Vector System |
| 4. |
Wang, L., Zheng, G.G., Ma, C.H., Lin, Y.M., Zhang, H.Y., Ma, Y.Y., Chong, J.H. and Wu, K.F.
(2008)
A special linker between macrophage and hematopoietic malignant cells: membrane form of macrophage colony-stimulating factor.
Cancer Res.
68
,
5639–5647
.
|
| |
Notes:
To examine the role of the membrane form of macrophage colony–stimulating factor(mM-CSF) in the hematopoietic system, RT-PCR was used amplify the cDNA of human mM-CSF from J6-1 cells, a human leukemia cell line. The PCR product was digested and cloned into the pTargeT™ Mammalian Expression Vector. After sequencing to verify the sequence, the construct and empty pTargeT™ Mammalian Expression Vector were purified and used to transfect Namalwa and Ramos cells, human Burkitt’s lymphoma cell lines, in 24-well plates. The transfected cells were then selected for stable expression of the transfected vector using 1.4 mg/ml G418. Expression of mM-CSF and neomycin (in the empty vector) was confirmed using RT-PCR. These cells were injected into mice and the oncogenicity of the cells determined using antibody staining of tissues.
(0003989) |
| |
|
| |
Products: pTARGET™ Mammalian Expression Vector System |
| 5. |
Lee, J., Yu, P., Xiao, X. and Kodadek, T.
(2008)
A general system for evaluating the efficiency of chromophore-assisted light inactivation (CALI) of proteins reveals Ru(II) tris-bipyridyl as an unusually efficient "warhead".
Mol. Biosyst.
4
,
59-65
.
|
| |
Notes:
In this paper, researchers were looking for efficient chromophores for singlet oxygen generation used for chromophore-assisted light inactivation (CALI) of proteins. The HaloTag® protein and firefly luciferase were used to test how well the chromophores performed in crude extracts and living cells. The expression vector for an epitope-tagged Luciferase-HTP protein, 3X Flag-Luc-HTP-Myc, was constructed using firefly luciferase amplified from the pGL3-Basic Vector and HaloTag® (HTP) amplified from the pHT2 Vector. The fusion protein was tested for labeling with a HaloTag® biotin ligand by transfecting HeLa cells with 8μg of 3X Flag-Luc-HTP-Myc plasmid and 80ng of pRL-SV40 Vector. After transfection, cells were lysed with Passive Lysis Buffer and 2μl of HeLa cell lysate was diluted in 48μl of PBS + BSA and incubated for 30 minutes at room temperature with increasing concentrations of a biotin-HT ligand. The samples then were incubated with streptavidin-agarose for 30 minutes at room temperature, centrifuged and the luciferase activity of 20μl of supernatant was measured using the Dual-Luciferase® Reporter Assay System. The fusion protein was also tested using two chromophore ligands, ruthenium(II)tris-bipyridyl (Ru-HaloTag®[HT]) and fluoroscein-HT at a concentration of 100nM, and both were successful as measured by the Dual-Luciferase® Reporter Assay System. An in vivo CALI was performed by transfecting HeLa cells with 100ng of 3X Flag-HTP-Luc-Myc plasmid and 1ng of pRL-SV40 Vector for 15 hours, and treating the cells with Ru-HT or F-HT for 3 hours. The cells were then irradiated for 30 minutes, placed in the dark for 30 minutes, then the cells were lysed and analyzed with the DLR Assay.
(0003954) |
| |
|
| |
Products: Dual-Luciferase® Reporter 1000 Assay System | Dual-Luciferase® Reporter Assay System | Dual-Luciferase® Reporter Assay System 10-Pack | HaloTag® pHT2 Vector | pGL3-Basic Vector | pRL-SV40 Vector |
| 6. |
Takahashi, T., Murakami, K., Nagakura, M., Kishita, H., Watanabe, S., Honke, K., Ogura, K., Tai, T., Kawasaki, K., Miyamoto, D., Hidari, K.I., Guo, C.T., Suzuki, Y. and Suzuki, T.
(2008)
Sulfatide is required for efficient replication of influenza A virus.
J. Virol.
12
,
5940–50
.
|
| |
Notes:
Sulfatide is present in mammalian organs where influenza A replicates. Ceramide galactosyltransferase (CGT) and cerebroside (galactosylceramide) sulfotransferase (CST), which synthesize sulfatide, were cloned by PCR into the pTargeT™ Mammalian Expression Vector and the pGEM®-T Easy Vector, (CST with or without a three base insertion), respectively. The two genes were removed by restriction digestion and cloned into pIRES-neo to forma bicistronic construct. Arylsulfatase A (ASA), which degrades sulfatide was also amplified and cloned into the pGEM®-T Easy Vector, before being sublconed into a neomycin-resistant expression vector. The expression vectors were transfected into COS-7 cells and selected for stable expression using G418.
(0003990) |
| |
|
| |
Products: Antibiotic G-418 Sulfate | Antibiotic G-418 Sulfate Solution | pGEM®-T Easy Vector System I | pGEM®-T Easy Vector System II | pTARGET™ Mammalian Expression Vector System |
| 7. |
Lee, B.C., Le, D.T. and Gladyshev, V.N.
(2008)
Mammals reduce methionine-S-sulfoxide with MsrA and are unable to reduce methionine-R-sulfoxide, and this function can be restored with a yeast reductase.
J. Biol. Chem.
283
,
28361–28369
.
|
| |
Notes:
The C-terminus of free methionine-R-sulfoxide reductase (fRMsr) gene, a yeast gene that can generate methionine from methionine-S-sulfoxide and methionine-R-sulfoxide, was tagged with six histidines before being cloned into the pCI-neo Mammalian Expression Vector. This construct was transfected into SK-Hep1 hepatocytes with or without the Clonegene pEGFP-N1 Vector. Using 800 μg/ml G418 sulfate, a stable cell line was selected. This cell line was then tested for response to oxidative stress.
(0003985) |
| |
|
| |
Products: pCI-neo Mammalian Expression Vector |
| 8. |
Hou, Q., Wu, Y.H., Grabsch, H., Zhu, Y., Leong, S.H., Ganesan, K., Cross, D., Tan, L.K., Tao, J., Gopalakrishnan, V., Tang, B.L., Kon, O.L. and Tan, P.
(2008)
Integrative genomics identifies RAB23 as an invasion mediator gene in diffuse-type gastric cancer.
Cancer Res.
68
,
4623–4630
.
|
| |
Notes:
In this article, the researchers explored the role of RAB23 in gastric cancers. Twenty-four hours before transfection, AGS cells (gastric cancer cell line) that expressed RAB23 were seeded into a 24-well plate at a density of 1.8 × 105 cells/ml. To overexpress RAB23, a full-length RAB23 cDNA was cloned into the pCI-neo Mammalian Expression Vector and transfected into AGS cells. The effect of RAB23 overexpression on the cells was determined using a Matrigel invasion assay while protein expression levels were visualized with Western blotting.
(0003986) |
| |
|
| |
Products: pCI-neo Mammalian Expression Vector |
| 9. |
Seabold, G.K., Wang, P.Y., Chang, K., Wang, C.Y., Wang, Y.X., Petralia, R.S. and Wenthold, R.J.
(2008)
The SALM family of adhesion-like molecules forms heteromeric and homomeric complexes.
J. Biol. Chem.
283
,
8395-8405
.
|
| |
Notes:
To study the potential interactions among the five SALMs (synaptic adhesion-like molecules) family members, the cDNAs of the five SALMs were subcloned and then transiently transfected in various combinations of two to five SALMs into HEK293 cells. The cells were transfected using calcium phosphate precipitation with an equimolar amount of the pAdVAntage™ Vector to enhance protein production. Interactions of the expressed SALMs were analyzed by immunoprecipitation experiments.
(0003754) |
| |
|
| |
Products: pAdVAntage™ Vector |
| 10. |
Heikkinen, L.S., Kazlauskas, A., Melén, K., Wagner, R., Ziegler, T., Julkunen, I. and Saksela, K.
(2008)
Avian and 1918 Spanish influenza a virus NS1 proteins bind to Crk/CrkL Src homology 3 domains to activate host cell signaling.
J. Biol. Chem.
283
,
5719–27
.
|
| |
Notes:
The authors identified the N-terminal Src homology 3 (SH3) domain-binding motif of Crk and CrkL as the preferred binding partner of nonstructural protein 1 (NS1), an important virulence factor of the influenza A virus. Interaction of NS1 with Crk, CrkL and other SH3 domain-containing proteins p85, p85β and Eps8L1 was investigated by protein pull-down assays. Expression constructs for biotinylated Crk, CrkL, p85, p85β and Eps8L1 were created by amplifying the 123 amino acid biotin acceptor domain from Propionibacterium shermanii from the PinPoint Xa-T Vector and inserting it upstream of the protein-coding sequences in a pGEX vector derivative. These constructs and a construct encoding Myc-tagged NS1 were transfecting into 293FT cells, and the biotinylated proteins were immobilized from the cell lysate using TetraLink™ Tetrameric Avidin Resin. Any Myc-NS1 that bound to the immobilized protein was detected using Western Blot analysis and an anti-Myc antibody. The authors also investigated the ability of wildtype NS1 or NS1 mutants to inhibit interferon-induced gene expression. A reporter plasmid was created by cloning an interferon-stimulated response element upstream of a minimal thymidine kinase promoter driving firefly luciferase expression. A vector containing Renilla luciferase was used as a control to normalize transfection efficiency. Huh-7 cells were cotransfected with the firefly and Renilla luciferase reporter constructs (0.2µg and 5ng, respectively), treated with interferon-β and lysed using the Passive Lysis Buffer. Firefly and Renilla luciferase activities were measured using the Dual Luciferase Reporter Assay System.
(0003803) |
| |
|
| |
Products: Dual-Luciferase® Reporter Assay System | TetraLink™ Tetrameric Avidin Resin |
| 11. |
Tao, R.H. and Maruyama, I.N.
(2008)
All EGF(ErbB) receptors have preformed homo- and heterodimeric structures in living cells.
J. Cell Sci.
121
,
3207–3217
.
|
| |
Notes:
The CheckMate™ Mammalian Two-Hybrid System was used to explore the dimerization of epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases without ligand. The intracellular domains of EGFR, ErbB2, ErbB3 and ErbB4 were amplified and cloned into both the pACT and pBIND Vectors. Transfection into NIH3T3 cells in 12-well plates occurred with 0.3 μg of pG5luc Vector (the reporter vector), 0.2 μg of pACT Vector or an equimolar amount of pACT construct, and 0.1 μg of pBIND Vector or an equimolar amount of pBIND construct. After 24 hours, the cells were lysed and luciferase activity assessed using the Dual-Luciferase® Reporter Assay System.
(0003993) |
| |
|
| |
Products: CheckMate™ Mammalian Two-Hybrid System | Dual-Luciferase® Reporter 1000 Assay System | Dual-Luciferase® Reporter Assay System | Dual-Luciferase® Reporter Assay System 10-Pack |
| 12. |
Lin, H., Lee, E., Hestir, K., Leo, C., Huang, M., Bosch, E., Halenbeck, R., Wu, G., Zhou, A., Behrens, D., Hollenboguh, D., Linnemann, T., Qin, M., Wong, J., Chu, K., Doberstein, S.K. and Williams, L.T.
(2008)
Discovery of a cytokine and its receptor by functional screening of the extracellular proteome.
Science
320
,
807-11
.
|
| |
Notes:
The authors of this study created a cDNA library representative of the extracellular proteome (secreted proteins and the extracellular domains of transmembrane proteins). Each cDNA was individually transfected into 293T cells. Medium from the cDNA of each transfection was used in a suite of cell-based assays. The CellTiter-Glo® Assay was used to screen for secreted factors from the cell lines expressing the cDNA that affected viability of twelve cell lines: human primary B cells, human primary T cells, human primary NK cells, human primary monocytes, A549 cells, Colo205 cells, U-118 cells, MDA-MB231 cells, PC3 cells, PANC1 cells, human primary skeletal muscle progenitor cells, and rat primary oligodendrocyte precursor cells.
(0003935) |
| |
|
| |
Products: CellTiter-Glo® Luminescent Cell Viability Assay |
| 13. |
Miao, Q., Sun, Y., Wei, T., Zhao, X., Zhao, K., Yan, L., Zhang, X., Shu, H. and Yang, F.
(2008)
Chymotrypsin B cached in rat liver lysosomes and involved in apoptotic regulation through a mitochondrial pathway.
J. Biol. Chem.
283
,
8218–28
.
|
| |
Notes:
The authors characterized a novel caspase 8-like activity that cleaves Bid and activates the mitochondrial apoptotic pathway. This activity was purified from rat liver lysosomal extracts and later identified as chymotrypsin B (CtrB). CtrB was previously thought to be expressed only in the pancreas, but the authors were able to detect crtB RNA in total RNA from primary rat hepatocytes and a rat hepatoma cell line (RH-35) using RT-PCR and the Access RT-PCR System. To confirm the intralysosomal localization of Crt B, the authors transfected RH-35 cells with an expression vector encoding CrtB tagged with green fluorescent protein. Prior to transfection, synthesis of functional protein from the expression vector was confirmed by in vitro transcription and translation using the TNT® Coupled Transcription Translation System and [35S] methionine.
(0003889) |
| |
|
| |
Products: Access RT-PCR System | TNT® T7 Coupled Reticulocyte Lysate System |
| 14. |
Zhang, X., Horrell, S.A., Delaney, D., Gottlieb, D.I.
(2008)
Embryonic Stem Cells as a Platform for Analyzing Neural Gene Transcription
Stem Cells
26
,
1841-1849
.
|
| |
Notes:
The authors note that while spatially and temporally specific gene transcription is a fundamental process in the normal development of mammalian stem cells, transcription in stem cells is currently studied by a set of methodologies with significant limitations. For instance, transient transfections analyze gene regulatory elements in nonchromosomal context. Using transgenic mice places transgenes in chromosomal context, however the chromosomal site where the transgene is inserted strongly influences the transgenes expression. As well, the need to make transgenic mice limits the number of experiments that can be done. ESCs can overcome these limitation. Undifferentiated stem cells are suitable for genetic engineering approaches such as gene targeting and recombinase-mediated cassette exchanges. By using such techniques, precisely planned alteration of native genes such as insertion of reporters, deletions of nearby or distant DNA sequences and mutational substitutions can be made. The authors wanted to analyze the Olig2 gene, a helix-loop-helix transcription factor expressed in the developing nervous system. Because Olig2 plays a central role in differentiation, understanding how it is regulated is important to understanding the larger transcriptional network controling development.
To this end, the authors used vectors for transient transfection experiments, constructed by amplifying regions of the Olig2 gene by PCR using primers tailed with appropriate restrictions sites and cloning the fragments into a pGL3 Luciferase Reporter Vector (Cat. # E1741, e1751, e1761, e1771). Promoter-reporter DNA was transfected into ESCs, cells were cultured 24 hours, then luciferase assays (Promega, type not specified) used to measure transgene expression.
(0003918) |
| |
|
| |
|
| 15. |
Falk, A., Karlsson, TE, Kurdija, S., Frisen, J., Zupicich, J.
(2007)
High-throughput identification of genes promoting neuron formation and lineage choice in mouse embryonic stem cells
Stem Cells
25
,
1539-1545
.
|
| |
Notes:
Heterogeneous culture maturation over several days provided an opportunity to influence several different steps in the differentiation process from ES to mature, post-mitotic cells. The authors procured a mouse genomic committee rearrayed IRAV library of approximately 8,000 full-length mouse cDNAs in an expression vector under the CMV promoter. For an easy and efficient gene delivery method, they used liposome-based transfection and were able to reliably achieve greater than 80% transfection efficiency of monolayer cultures as detected by flow cytometry for CMV-eGFP expression. This high rate of transfection suggests that differentiating cells were accessible over the course of at least 4 days to exogenously expressed genes from plasmid DNA. DNA isolation was accomplished using PureYield™ Midiprep System (Cat. A2492).
(0003921) |
| |
|
| |
Products: PureYield™ Plasmid Midiprep System |
| 16. |
Tan, K.P., Yang, M. and Ito, S.
(2007)
Activation of Nrf2 by toxic bile acids provokes adaptive defense responses to enhance cell survival at the emergence of oxidative stress.
Mol. Pharmacol.
72
,
1380–1390
.
|
| |
Notes:
The authors explored the role that nuclear factor (erythroid 2-like) factor 2 (Nrf2) may have in mitigating the cytotoxic effects of bile acids on cells. A reporter vector was constructed using the core sequence of antioxidant reporter element (ARE) sythnesized by annealing two complementary oligonucleotides with Kpn1 and BglII at the 5’ and 3’ ends, respectively, and ligated into the same restriction sites of the pGL3-Promoter Vector (designated pGL3_ARE). To ensure specificity for the experiments, three point mutations were introduced into the ARE sequence (designated pGL3_mARE). To create HepG2 cells that stably expressed human Na(+)-taurocholate co-transporting polypeptide (NTCP), the cDNA clone of NTCP was subcloned into pTargeT™ Mammalian Expression Vector via the NotI site and selected using 500μg/ml G-418. The stable clones or standard HepG2 cells were transiently transfected with 0.1µg of pGL3_ARE or pGL3_mARE, 0.02µg of the control reporter pRL-TK Vector, with or without Nrf2 or dominant negative Nrf2 expression constructs. After overnight transfection, the cells were treated with bile acids for 16–18 hours and luciferase activities determined using the Dual-Luciferase® Reporter Assay System. Each experiment was done in triplicate and repeated at least two times.
(0003691) |
| |
|
| |
Products: Dual-Luciferase® Reporter 1000 Assay System | Dual-Luciferase® Reporter Assay System | Dual-Luciferase® Reporter Assay System 10-Pack | pGL3-Promoter Vector | pRL-TK Vector | pTARGET™ Mammalian Expression Vector System |
| 17. |
Nakajima, T., Hayashi, K., Viswanathan, P.C., Kim, M.Y., Anghelescu, M., Barksdale, K.A., Shuai, W., Balser, J.R. and Kupershmidt, S.
(2007)
HERG is protected from pharmacological block by alpha-1,2-glucosyltransferase function.
J. Biol. Chem.
282
,
5506-5513
.
|
| |
Notes:
The pSI Mammalian Expression Vector was used to subclone HERG (human ether-à-go-go-related gene) cDNA and then subjected to site-directed mutagenesis. The constructs were used for transient transfection and tested for membrane current.
(0003756) |
| |
|
| |
Products: pSI Mammalian Expression Vector |
| 18. |
Zhang, Y-w., Wang, R., Liu, Q., Zhang, H., Liao, F-F. and Xu, H.
(2007)
Presenilin/γ-secretase-dependent processing of β-amyloid precursor protein regulates EGF receptor expression
Proc. Natl. Acad. Sci. U S A
104
,
10613-10618
.
|
| |
Notes:
The authors of this study investigated the downstream effects of the release of the intracellular domain of the Amyloid-β precursor protein (AICD) on cellular activities. They amplified the 5′ region of the mouse EGFR gene and cloned it into a pGL3 vector. This construct was cotransfected into embryonic fibroblasts derived from APP/APLP2 DKO mice along with a vector expressing AICD, AICD and the multidomain protein Fe65, Fe65 alone or NotchΔE, along with a Renilla control vector to normalize data for transfection efficiency. The data indicate that AICD negatively regulates transcription of the EGF Receptor gene.
(0003861) |
| |
|
| |
Products: pGL3-Promoter Vector | pRL-SV40 Vector |
| 19. |
Boggs, K. and Reisman, D.
(2007)
C/EBPbeta participates in regulating transcription of the p53 gene in response to mitogen stimulation.
J. Biol. Chem.
282
,
7982–7890
.
|
| |
Notes:
To explore the role of C/EBPb isoforms in regulating p53 expression during the cell cycle, the 1.7 kb murine p53 promoter was cloned into the pGL3-Basic Vector. Using TransFast™ Reagent, Swiss3T3 and 6629 (C/EBPb-null) cells were transfected with 0.1–0.75 µg of pGL3-1.7-kb p53 promoter construct with or without co-transfection of 0.25 µg of C/EBPb-2, and with 50 ng of pRL-TK Vector as an internal control. Twenty-four hours post-transfection, the cells were harvested and assayed for luciferase activity, normalizing reporter activity to Renilla luciferase activity. The GeneEditor™ in vitro Site-Directed Mutagenesis System was used to either mutate or delete the –972/–953 cis-acting element carrying the C/EBPb-binding site within the p53 promoter, and 0.1–0.75 µg of the mutant constructs were then tested in the same reporter assay. The C/EBPb-1, -2, and -3 cDNAs were cloned into an expression vector and translated using 0.5µg of plasmid in the TNT® T7 Quick Coupled transcription/translation system either in the presence of unlabeled or [35S]methionine. The synthesized proteins were analyzed on 12% SDS-polyacrylamide gel.
(0003675) |
| |
|
| |
Products: GeneEditor™ in vitro Site-Directed Mutagenesis System | pGL3-Basic Vector | pRL-TK Vector | TNT® T7 Quick Coupled Transcription/Translation System | TNT® T7 Quick Coupled Transcription/Translation System, Trial Size | TransFast™ Transfection Reagent |
| 20. |
Igarashi, M., Yogiashi, Y., Mihara, M., Takada, I., Kitagawa, H. and Kato, S.
(2007)
Vitamin K induces osteoblast differentiation through pregnane X receptor-mediated transcriptional control of the Msx2 gene.
Mol. Cell. Biol.
27
,
7947-7954
.
|
| |
Notes:
Igarashi et al. showed that expression of the Msx2 gene, which enodes a transcription factor, is induced by vitamin K treatment via a pregnane X receptor response element (PXRE) and by estrogen via an estrogen response element (ERE). Promoter analysis was performed by cloning the Msx2 promoters into the pGL3-Basic Vector, transfecting MC3T3 and ST2 cells with the pGL3-Basic constructs, treating the cell with 10nM 17β-estradiol and 10µM vitamin K, then measuring luciferase activity. The pRL-CMV Vector (2.5ng per well of a 12-well plate) was cotransfected to normalize for transfection efficiency. The ability of PXR to bind to the Msx2-PXRE was assessed by an avidin-biotin complex DNA assay. Sense and antisense oligonucleotides that were biotinylated at the 5´ end were annealed and immobilized with the TetraLink™ Tetrameric Avidin Resin. HEK293T cells were lysed with lysis buffer (10mM Tris-HCl [pH7.8], 1mM EDTA, 150mM NaCl, 0.1% NP-40) containing protease inhibitors, then centrifuged to clarify the extract. The supernatants were mixed with the DNA-TetraLink™ Resin to allow proteins to bind to the oligos, and resin was washed with lysis buffer. Bound proteins were analyzed by SDS-PAGE and Western blotting.
(0003805) |
| |
|
| |
Products: pGL3-Basic Vector | pRL-CMV Vector | TetraLink™ Tetrameric Avidin Resin |
| 21. |
Kanayama, T., Arito, M., So, K., Hachimura, S., Inoue, J. and Sato, R.
(2007)
Interaction between sterol regulatory element-binding proteins and liver receptor homolog-1 reciprocally suppresses their transcriptional activities.
J. Biol. Chem.
282
,
10290–10298
.
|
| |
Notes:
To explore the interaction of liver receptor homolog (LRH)-1, a known suppressor of sterol regulatory element-binding protein (SREBP) transcriptional activity, human LRH-1 was reverse transcribed then amplified by PCR from total RNA from HepG2 cells. The amplification product was ligated into the pTargeT™ Mammalian Expression Vector to create pTarget-LRH1. For reporter experiments, a PCR fragment that encompassed the 1.3kb 5’-promoter region of the human small heterodimer partner (SHP) gene was cloned into the pGL3-Basic Vector (designated pSRB). The pGL3-Promoter Vector was used to construct pLRHREx3, which contains three LRH-1 response elements, and the insert was generated using synthetic oligonucleotides. HEK293 cells were cotransfected with 0.2µg of a promoter-firefly luciferase construct, 0.1µg of a SREBP expression plasmid, 10ng of phRL-TK Vector and 0.2 or 0.6µg of pTarget-LRH1. Alternatively, the cotransfected plasmids were 0.2µg of pSHP, 0.1µg of pTarget-LRH1, 10ng of phRL-TK Vector and 0.2 or 0.6µg of a SREBP expression plasmid. The pLRHREx3 construct (0.2µg) was cotransfected with 0.1µg of a LRH-1 expression plasmid, 0.2µg of pCMXPGC-1α (peroxisome proliferator activated receptor γ coactivator-1α), 10ng of phRL-TK Vector, and 0.1 or 0.3µg of a pSREBP expression vector in HEK 293 cells. Luciferase expression was assayed 48 hours post-transfection using the Dual-Luciferase® Assay Reporter System. To express SREBPs and LRH-1 in vitro, inserts were ligated into the pTNT™ Vector, synthesized using the TNT® Coupled Transcription/Translation System with radiolabeled methionine. Ten microliters of the 35S-labelled protein was then used in a GST-pulldown assay.
(0003692) |
| |
|
| |
Products: Dual-Luciferase® Reporter 1000 Assay System | Dual-Luciferase® Reporter Assay System | Dual-Luciferase® Reporter Assay System 10-Pack | pGL3-Basic Vector | pGL3-Promoter Vector | pTNT™ Vector | TNT® SP6 Coupled Reticulocyte Lysate System | TNT® SP6 Coupled Reticulocyte Lysate System, Trial Size | TNT® T3 Coupled Reticulocyte Lysate System | TNT® T7 Coupled Reticulocyte Lysate System | TNT® T7 Coupled Reticulocyte Lysate System, Trial Size |
| 22. |
Schäfer, P., Cymerman, I.A., Bujnicki, J.M. and Meiss, G.
(2007)
Human lysosomal DNase IIα contains two requisite PLD-signature (HxK) motifs: evidence for a pseudodimeric structure of the active enzyme species.
Protein Sci.
16
,
82–91
.
|
| |
Notes:
The authors used biochemical and mutational analysis to characterize human lysosomal DNaseIIα. Native DNaseIIα and site-directed mutants were expressed as Flag-His6-DNaseIIα and HA-tagged DNaseIIα using expression constructs created with the pCI Vector. Protein was expressed by transiently transfecting HEK 293-T cells using the TransFast™ Transfection Reagent. Flag-His6-DNaseIIα was purified using the MagneHis™ Ni-Particles, and this purified protein was used in nuclease assays to monitor catalytic activity and in gel filtration experiments and coimmunoprecipitation assays with HA-DNaseIIα to determine whether the active enzyme is monomeric.
(0003786) |
| |
|
| |
Products: MagneHis™ Ni-Particles | pCI Mammalian Expression Vector | TransFast™ Transfection Reagent |
| 23. |
Zhuang, H. and Matsunami, H.
(2007)
Synergism of accessory factors in functional expression of mammalian odorant receptors.
J. Biol. Chem.
282
,
15284–15293
.
|
| |
Notes:
To explore the role that accessory proteins may play in successfully expressing odorant receptors (ORs) on the cell surface of heterologous cells, the authors explored cotransfecting receptor-transporting proteins RTP1S, Ric8b and Gαolf with an N-terminally tagged OR to generate a functional and ligand-specific expression system. Three tags (Rho, FLAG or HA) were inserted into the NheI and EcoRI sites of the pCI Mammalian Expression Vector. Amplified OR orfs were cloned into the MluI and NotI sites of the tagged pCI Mammalian Expression Vector. The accessory proteins were subcloned into HA-pCI (RTP1S) or pCI (RTP1S, Ric8b, Hsc70t and Gαolf). For cotransfection, the N-terminal tagged OR vectors (0.8µg) and the assessory proteins (individually or in combination; 0.8µg) were transfected into HEK293T or Hana3A cells. The transfected cells were then subjected to immunocytochemistry, live-cell surface staining, permeabilized staining or FACS analysis. The Dual-Glo™ Luciferase Assay System was used to assess OR activation via CRE elements on a firefly luciferase vector. pRL-SV40 Vector was the internal control for cell viability and transfection efficiency. HEK293T or Hana3A cells were plated on 96-well plates, transfected with 1µg of CRE-Luc vector, 1µg of pRL-SV40 Vector, 5µg of OR and 1µg total for all accessory proteins (0.25µg each protein with pCI Mammalian Expression Vector to keep amount of plasmid constant). Twenty-four hours posttransfection, the medium was changed to CD293 chemically defined medium, incubated for 30 minutes at 37°C then replaced with 25µl of odorant solution in CD293 for a second incubation of 4 hours at 4°C. Then the reporter protein expression levels were measured by luminescence.
(0003687) |
| |
|
| |
Products: Dual-Glo® Luciferase Assay System | pCI Mammalian Expression Vector | pRL-SV40 Vector |
| 24. |
Gubser, C., Goodbody, R., Ecker, A., Brady, G., O'Neill, L.A., Jacobs, N. and Smith, G.L.
(2007)
Camelpox virus encodes a schlafen-like protein that affects orthopoxvirus virulence.
J. Gen. Virol.
88
,
1667-1676
.
|
| |
Notes:
The authors wanted to examine how the Camelpox virus (CMLV) gene 176R (v-slfn) might affect orthopoxvirus virulence since it shares sequence similarity to murine schlafen (m-slfn) proteins that inhibit T cell and fibroblast growth. HeLa cells were transiently transfected with either pCI Mammalian Expression Vector alone or pCI Mammalian Expression Vector expressing v-slfn with or without a HA tag. Cellular localization of the protein was visualized using either α-v-slfn antiserum or α-HA mAb.
(0003757) |
| |
|
| |
Products: pCI Mammalian Expression Vector |
| 25. |
Sullivan, C., Postlethwait, J.H., Lage, C.R., Millard, P.J. and Kim, C.H.
(2007)
Evidence for evolving Toll-IL-1 receptor-containing adaptor molecule function in vertebrates.
J. Immunol.
178
,
4517-4527
.
|
| |
Notes:
The authors used reporter assays and coimmunoprecipitation experiments to compare the zebrafish (Danio rerio) Toll-IL-1R-containing adaptor molecule 1 (TICAM1) activation of NF-κB and zebrafish type I IFN to mammalian TICAM1 activation. 293H and ZFL (zebrafish liver) cells were cotransfected with 400 ng of TICAM1 construct (mouse, zebrafish or a deletion construct), 400 ng of a reporter construct (e.g., a zebrafish IFN promoter cloned into the pGL3-Basic Vector) and 10 ng of pRL-CMV Vector, an internal control to normalize data. After 24 hours for 293H cells and 48 hours for ZFL cells, the transfected cells were lysed, and luciferase activity was measured using the Dual-Luciferase® Reporter Assay System. For coimmunoprecipitation experiments, 293H cells were cotransfected with a total of 8µg of plasmids (3µg of zebrafish or mouse TICAM1 construct, 3µg of interacting protein construct, 1µg of the pAdVAntage™ Vector, and 1µg of antiapoptotic protein p35 construct). Forty-eight hours posttransfection, the cells were lysed, the protein bound to affinity resins and analyzed by Western blot.
(0003755) |
| |
|
| |
Products: Dual-Luciferase® Reporter Assay System | pAdVAntage™ Vector | pGL3-Basic Vector | pRL-CMV Vector |