Combined RNAi-Mediated Suppression of Rictor and EGFR Resulted in Complete Tumor Regression in an Orthotopic Glioblastoma Tumor Model
The PI3K/AKT/mTOR pathway is commonly over activated in glioblastoma (GBM),and Rictor was shown to be an important regulator downstream of this pathway. EGFR overexpression is also frequently found in GBM tumors,and both EGFR and Rictor are associated with increased proliferation,invasion,metastasis and poor prognosis. This research evaluated in vitro and in vivo whether the combined silencing of EGFR and Rictor would result in therapeutic benefits. The therapeutic potential of targeting these proteins in combination with conventional agents with proven activity in GBM patients was also assessed. In vitro validation studies were carried out using siRNA-based gene silencing methods in a panel of three commercially available human GBM cell lines,including two PTEN mutant lines (U251MG and U118MG) and one PTEN-wild type line (LN229). The impact of EGFR and/or Rictor silencing on cell migration and sensitivity to chemotherapeutic drugs in vitro was determined. In vivo validation of these studies was focused on EGFR and/or Rictor silencing achieved using doxycycline-inducible shRNA-expressing U251MG cells implanted orthotopically in Rag2M mice brains. Target silencing,tumor size and tumor cell proliferation were assessed by quantification of immunohistofluorescence-stained markers. siRNA-mediated silencing of EGFR and Rictor reduced U251MG cell migration and increased sensitivity of the cells to irinotecan,temozolomide and vincristine. In LN229,co-silencing of EGFR and Rictor resulted in reduced cell migration,and increased sensitivity to vincristine and temozolomide. In U118MG,silencing of Rictor alone was sufficient to increase this line's sensitivity to vincristine and temozolomide. In vivo,while the silencing of EGFR or Rictor alone had no significant effect on U251MG tumor growth,silencing of EGFR and Rictor together resulted in a complete eradication of tumors. These data suggest that the combined silencing of EGFR and Rictor should be an effective means of treating GBM.
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产品类型：
产品号#：
05750
05751
产品名：
NeuroCult™ NS-A 基础培养基（人）
NeuroCult™ NS-A 扩增试剂盒（人）
Park A et al. ( 2014)
PloS one 9 4 e95101
CRISPR/Cas9 allows efficient and complete knock-in of a destabilization domain-tagged essential protein in a human cell line, allowing rapid knockdown of protein function.
Although modulation of protein levels is an important tool for study of protein function,it is difficult or impossible to knockdown or knockout genes that are critical for cell growth or viability. For such genes,a conditional knockdown approach would be valuable. The FKBP protein-based destabilization domain (DD)-tagging approach,which confers instability to the tagged protein in the absence of the compound Shield-1,has been shown to provide rapid control of protein levels determined by Shield-1 concentration. Although a strategy to knock-in DD-tagged protein at the endogenous loci has been employed in certain parasite studies,partly due to the relative ease of knock-in as a result of their mostly haploid lifecycles,this strategy has not been demonstrated in diploid or hyperploid mammalian cells due to the relative difficulty of achieving complete knock-in in all alleles. The recent advent of CRISPR/Cas9 homing endonuclease-mediated targeted genome cleavage has been shown to allow highly efficient homologous recombination at the targeted locus. We therefore assessed the feasibility of using CRISPR/Cas9 to achieve complete knock-in to DD-tag the essential gene Treacher Collins-Franceschetti syndrome 1 (TCOF1) in human 293T cells. Using a double antibiotic selection strategy to select clones with at least two knock-in alleles,we obtained numerous complete knock-in clones within three weeks of initial transfection. DD-TCOF1 expression in the knock-in cells was Shield-1 concentration-dependent,and removal of Shield-1 resulted in destabilization of DD-TCOF1 over the course of hours. We further confirmed that the tagged TCOF1 retained the nucleolar localization of the wild-type untagged protein,and that destabilization of DD-TCOF1 resulted in impaired cell growth,as expected for a gene implicated in ribosome biogenesis. CRISPR/Cas9-mediated homologous recombination to completely knock-in a DD tag likely represents a generalizable and efficient strategy to achieve rapid modulation of protein levels in mammalian cells.
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产品类型：
产品号#：
73342
73344
产品名：
嘌呤霉素 (Dihydrochloride)
嘌呤霉素 (Dihydrochloride)
Khorashad JS et al. (JUN 2009)
Haematologica 94 6 861--4
The level of BCR-ABL1 kinase activity before treatment does not identify chronic myeloid leukemia patients who fail to achieve a complete cytogenetic response on imatinib.
Imatinib is currently the first line therapy for newly diagnosed patients with chronic myeloid leukemia. However,20-25% of patients do not achieve durable complete cytogenetic responses. The mechanism underlying this primary resistance is unknown,but variations in BCR-ABL1 kinase activity may play a role and can be investigated by measuring the autophosphorylation levels of BCR-ABL1 or of a surrogate target such as Crkl. In this study we used flow cytometry to investigate the in vitro inhibition of Crkl phosphorylation by imatinib in CD34(+) cells in diagnostic samples from two groups of patients distinguished by their cytogenetic response. No difference in inhibition of Crkl phosphorylation was observed in the two groups. The observation that increasing the dose of imatinib in vivo did not increase the level of cytogenetic response in some non-responders suggests that in at least a proportion of patients imatinib resistance may be due to activation of BCR-ABL1-independent pathway.
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产品类型：
产品号#：
18056
18056RF
产品名：
Ueno NT et al. (NOV 2003)
Blood 102 10 3829--36
Rapid induction of complete donor chimerism by the use of a reduced-intensity conditioning regimen composed of fludarabine and melphalan in allogeneic stem cell transplantation for metastatic solid tumors.
We evaluated the feasibility and efficacy of a reduced-intensity conditioning (RIC) regimen of fludarabine and melphalan to achieve rapid complete donor chimerism after allogeneic stem cell transplantation (SCT) in patients with metastatic solid tumors. Between January 1999 and January 2003,8 patients with metastatic breast cancer (BC) and 15 with metastatic renal cell carcinoma (RCC) underwent allogeneic SCT after an RIC regimen of 5 days of fludarabine and 2 days of melphalan. Filgrastim-mobilized stem cells from HLA-identical related or unrelated donors were infused. Prophylaxis for graft-versus-host disease (GVHD) consisted of tacrolimus and methotrexate. All 22 evaluable patients had 100% donor chimerism at day 30 and at all measurement times thereafter. One patient died 19 days after SCT. Nine patients (39%) had grades II to IV acute GVHD and 10 patients (43%) had chronic GVHD. Five patients (22%) died of nonrelapse treatment-related complications. Treatment-related disease response was seen in 10 patients (45%),with 3 complete responses,2 partial responses,and 5 minor responses. Fludarabine-melphalan is a feasible and effective RIC regimen for allogeneic SCT in metastatic BC and RCC. It induces rapid complete donor chimerism without the need for donor lymphocyte infusion. Tumor regression associated with GVHD is consistent with graft-versus-tumor effect.
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产品类型：
产品号#：
15271HLA
产品名：
RosetteSep™ HLA 淋系细胞富集试剂盒
Lannutti BJ et al. (FEB 2009)
Blood 113 8 1778--85
Incomplete restoration of Mpl expression in the mpl-/- mouse produces partial correction of the stem cell-repopulating defect and paradoxical thrombocytosis.
Expression of Mpl is restricted to hematopoietic cells in the megakaryocyte lineage and to undifferentiated progenitors,where it initiates critical cell survival and proliferation signals after stimulation by its ligand,thrombopoietin (TPO). As a result,a deficiency in Mpl function in patients with congenital amegakaryocytic thrombocytopenia (CAMT) and in mpl(-/-) mice produces profound thrombocytopenia and a severe stem cell-repopulating defect. Gene therapy has the potential to correct the hematopoietic defects of CAMT by ectopic gene expression that restores normal Mpl receptor activity. We rescued the mpl(-/-) mouse with a transgenic vector expressing mpl from the promoter elements of the 2-kb region of DNA just proximal to the natural gene start site. Transgene rescued mice exhibit thrombocytosis but only partial correction of the stem cell defect. Furthermore,they show very low-level expression of Mpl on platelets and megakaryocytes,and the transgene-rescued megakaryocytes exhibit diminished TPO-dependent kinase phosphorylation and reduced platelet production in bone marrow chimeras. Thrombocytosis is an unexpected consequence of reduced Mpl expression and activity. However,impaired TPO homeostasis in the transgene-rescued mice produces elevated plasma TPO levels,which serves as an unchecked stimulus to drive the observed excessive megakaryocytopoiesis.
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Nagaoka M et al. (JAN 2010)
BMC developmental biology 10 60
Culture of human pluripotent stem cells using completely defined conditions on a recombinant E-cadherin substratum.
BACKGROUND: To maintain pluripotency of human embryonic stem (huES) cells in feeder-free culture it has been necessary to provide a Matrigel substratum,which is a complex of poorly defined extracellular matrices and growth factors derived from mouse Engelbreth-Holm-Swarm sarcoma cells. Culture of stem cells under ill-defined conditions can inhibit the effectiveness of maintaining cells in a pluripotent state and reduce reproducibility of differentiation protocols. Moreover recent batches of Matrigel have been found to be contaminated with the single stranded RNA virus,Lactate Dehydrogenase Elevating Virus (LDEV),raising concerns regarding the safety of using stem cells that have been cultured on Matrigel in a therapeutic setting. To circumvent such concerns,we attempted to identify a recombinant matrix that could be used as an alternative to Matrigel for the culture of human pluripotent stem cells. huES and human induced pluripotent stem (hiPS) cells were grown on plates coated with a fusion protein consisting of E-cadherin and the IgG Fc domain using mTeSR1 medium.backslashnbackslashnRESULTS: Cells grown under these conditions maintained similar morphology and growth rate to those grown on Matrigel and retained all pluripotent stem cell features,including an ability to differentiate into multiple cell lineages in teratoma assays. We,therefore,present a culture system that maintains the pluripotency of huES and hiPS cells under completely defined conditions.backslashnbackslashnCONCLUSIONS: We propose that this system should facilitate growth of stem cells using good manufacturing practices (GMP),which will be necessary for the clinical use of pluripotent stem cells and their derivatives.
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产品类型：
产品号#：
05850
05857
05870
05875
85850
85857
85870
85875
产品名：
mTeSR™1
mTeSR™1
Yang H et al. (JAN 2011)
Proceedings of the National Academy of Sciences of the United States of America 108 1 12--7
Completely phased genome sequencing through chromosome sorting.
The two haploid genome sequences that a person inherits from the two parents represent the most fundamentally useful type of genetic information for the study of heritable diseases and the development of personalized medicine. Because of the difficulty in obtaining long-range phase information,current sequencing methods are unable to provide this information. Here,we introduce and show feasibility of a scalable approach capable of generating genomic sequences completely phased across the entire chromosome.
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产品类型：
产品号#：
15705
产品名：
RosetteSep™DM-L密度梯度离心液
Liu C et al. (DEC 2010)
Blood 116 25 5518--27
Progenitor cell dose determines the pace and completeness of engraftment in a xenograft model for cord blood transplantation.
Two critical concerns in clinical cord blood transplantation are the initial time to engraftment and the subsequent restoration of immune function. These studies measured the impact of progenitor cell dose on both the pace and strength of hematopoietic reconstitution by transplanting nonobese diabetic/severe combined immunodeficiency/interleukin-2 receptor-gamma-null (NSγ) mice with lineage-depleted aldehyde dehydrogenase-bright CD34(+) human cord blood progenitors. The progress of each transplant was monitored over an extended time course by repeatedly analyzing the peripheral blood for human hematopoietic cells. In vivo human hematopoietic development was complete. After long-term transplantation assays (≥ 19 weeks),human T-cell development was documented within multiple tissues in 16 of 32 NSγ mice. Human T-cell differentiation was active within NSγ thymuses,as documented by the presence of CD4(+) CD8(+) T-cell progenitors as well as T-cell receptor excision circles. It is important to note that although myeloid and B-cell engraftment was detected as early as 4 weeks after transplantation,human T-cell development was exclusively late onset. High progenitor cell doses were associated with a robust human hematopoietic chimerism that accelerated both initial time to engraftment and subsequent T-cell development. At lower progenitor cell doses,the chimerism was weak and the human hematopoietic lineage development was frequently incomplete.
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产品类型：
产品号#：
01700
01705
01701
01702
14056
14066
28600
19056
19056RF
19756
19756RF
产品名：
ALDEFLUOR™ 试剂盒
ALDEFLUOR™ DEAB试剂, 1.5 mM, 1 mL
ALDEFLUOR™检测缓冲液
L-Calc™有限稀释软件
Baran-Marszak F et al. (DEC 2010)
Blood 116 26 5961--71
Expression level and differential JAK2-V617F-binding of the adaptor protein Lnk regulates JAK2-mediated signals in myeloproliferative neoplasms.
Activating mutations in signaling molecules,such as JAK2-V617F,have been associated with myeloproliferative neoplasms (MPNs). Mice lacking the inhibitory adaptor protein Lnk display deregulation of thrombopoietin/thrombopoietin receptor signaling pathways and exhibit similar myeloproliferative characteristics to those found in MPN patients,suggesting a role for Lnk in the molecular pathogenesis of these diseases. Here,we showed that LNK levels are up-regulated and correlate with an increase in the JAK2-V617F mutant allele burden in MPN patients. Using megakaryocytic cells,we demonstrated that Lnk expression is regulated by the TPO-signaling pathway,thus indicating an important negative control loop in these cells. Analysis of platelets derived from MPN patients and megakaryocytic cell lines showed that Lnk can interact with JAK2-WT and V617F through its SH2 domain,but also through an unrevealed JAK2-binding site within its N-terminal region. In addition,the presence of the V617F mutation causes a tighter association with Lnk. Finally,we found that the expression level of the Lnk protein can modulate JAK2-V617F-dependent cell proliferation and that its different domains contribute to the inhibition of multilineage and megakaryocytic progenitor cell growth in vitro. Together,our results indicate that changes in Lnk expression and JAK2-V617F-binding regulate JAK2-mediated signals in MPNs.
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产品类型：
产品号#：
03434
03444
04960
04902
04900
产品名：
MethoCult™ GF M3434
MethoCult™ GF M3434
MegaCult™-C胶原和无细胞因子培养基
胶原蛋白溶液
MegaCult™-C无细胞因子培养基
Hisa T et al. (JAN 2004)
The EMBO journal 23 2 450--9
Hematopoietic, angiogenic and eye defects in Meis1 mutant animals.
Meis1 and Hoxa9 expression is upregulated by retroviral integration in murine myeloid leukemias and in human leukemias carrying MLL translocations. Both genes also cooperate to induce leukemia in a mouse leukemia acceleration assay,which can be explained,in part,by their physical interaction with each other as well as the PBX family of homeodomain proteins. Here we show that Meis1-deficient embryos have partially duplicated retinas and smaller lenses than normal. They also fail to produce megakaryocytes,display extensive hemorrhaging,and die by embryonic day 14.5. In addition,Meis1-deficient embryos lack well-formed capillaries,although larger blood vessels are normal. Definitive myeloerythroid lineages are present in the mutant embryos,but the total numbers of colony-forming cells are dramatically reduced. Mutant fetal liver cells also fail to radioprotect lethally irradiated animals and they compete poorly in repopulation assays even though they can repopulate all hematopoietic lineages. These and other studies showing that Meis1 is expressed at high levels in hematopoietic stem cells (HSCs) suggest that Meis1 may also be required for the proliferation/self-renewal of the HSC.
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产品类型：
产品号#：
04960
04902
04900
04961
04901
04963
04962
04970
04971
产品名：
MegaCult™-C胶原和无细胞因子培养基
胶原蛋白溶液
MegaCult™-C无细胞因子培养基
MegaCult™-C胶原和含细胞因子培养基
MegaCult™-C含细胞因子培养基
双室载玻片套件
MegaCult™-C CFU-Mk染色试剂盒
MegaCult™-C无细胞因子全套试剂盒
MegaCult™-C含细胞因子全套试剂盒
Gekas C et al. (APR 2009)
Blood 113 15 3461--71
Mef2C is a lineage-restricted target of Scl/Tal1 and regulates megakaryopoiesis and B-cell homeostasis.
The basic helix-loop-helix transcription factor stem cell leukemia gene (Scl) is a master regulator for hematopoiesis essential for hematopoietic specification and proper differentiation of the erythroid and megakaryocyte lineages. However,the critical downstream targets of Scl remain undefined. Here,we identified a novel Scl target gene,transcription factor myocyte enhancer factor 2 C (Mef2C) from Scl(fl/fl) fetal liver progenitor cell lines. Analysis of Mef2C(-/-) embryos showed that Mef2C,in contrast to Scl,is not essential for specification into primitive or definitive hematopoietic lineages. However,adult VavCre(+)Mef2C(fl/fl) mice exhibited platelet defects similar to those observed in Scl-deficient mice. The platelet counts were reduced,whereas platelet size was increased and the platelet shape and granularity were altered. Furthermore,megakaryopoiesis was severely impaired in vitro. Chromatin immunoprecipitation microarray hybridization analysis revealed that Mef2C is directly regulated by Scl in megakaryocytic cells,but not in erythroid cells. In addition,an Scl-independent requirement for Mef2C in B-lymphoid homeostasis was observed in Mef2C-deficient mice,characterized as severe age-dependent reduction of specific B-cell progenitor populations reminiscent of premature aging. In summary,this work identifies Mef2C as an integral member of hematopoietic transcription factors with distinct upstream regulatory mechanisms and functional requirements in megakaryocyte and B-lymphoid lineages.
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EasySep™小鼠TIL（CD45）正选试剂盒
沪公网安备31010102008431号