pmRi-mCherry Vector

Description

The pmRi-mCherry Vector is a Tet-inducible mammalian expression vector that allows the coexpression of a user-generated microRNA miRNA and a red fluorescent protein, mCherry, to be controlled by a Tet System transactivator and doxycycline. The TRE-based promoter, PTight, regulates the expression of the mCherry mRNA transcript, which contains your miRNA precursor seq-uence embedded in its 3 UTR. Inducibility requires that a Tet System transactivator e.g. Tet-On Advanced also be expressed in the target cells. To select stable cell lines, the pmRi-mCherry Vector must be cotransfected with one of the provided linear selection markers. Our products are to be used forResearch Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval. Back Tetracycline-inducible microRNA.In the presence of doxycycline Dox , the Tet-On Advanced protein binds to the inducible promoter,PTight, and elicits high levels of transcription of a composite mRNA that encodes a fluorescent protein mCherry or ZsGreen1 and your miRNA sequence in its 3 UTR. Cells that fluoresce also express your microRNA. Back Tetracycline-inducible miRNA.The RNAiMonitor System was used to create vectors that express a luciferase reporter containing miR-1 or miR-9 cognate target sequences in their 3 UTRs 3 each . These reporters were then co-transfected with pmRi-ZsGreen1 vectors containing miR-1 or miR-9 miRNA inserts into MCF-7 Tet-On Advanced cells. The parent pmRi-ZsGreen1 vector provided a negative control for each experiment. Inducing miR-1 or miR-9 expression with Dox resulted in >90 % reduction in normalized luciferase activity Back Tetracycline-inducible shRNA in MCF-7 cells.MCF-7 Tet-On Advanced cells expressing an miR-1 miRNA construct from the pmRi-mCherry and pmRi-ZsGreen1 vectors, exhibit bright red and green fluorescence, respectively, but only in the presence of doxycycline. Back Required Products Fetal Bovine Serum FBS that has been functionally tested for optimal use with all of our Tet Systems. This FBS does not contain trace levels of tetracycline or derivatives which have been observed to interfere with proper regulation of TRE-controlled gene expression. Use of this serum ensures the maximal range of induction possible with the Tet Systems. This FBS has been processed in the United States. Back Fetal bovine serum FBS approved for use with tetracycline inducible expression systems.Only Takara Bio USA functionally tests FBS to be sure that no contaminating tetracycline derivatives interfere with induced expression levels. Back Fetal bovine serum from Clontech is functionally tested to guarantee consistent results with tetracycline-inducible expression systems.Serum from other vendors that have not been functionally tested may contain tetracyclines that affect the maximum expression in Tet-Off systems, or generate background in Tet-On systems. Data here shows induction of luciferase expression from our CHO-AA8-Luc Tet-Off Control Cell Line using different sources of FBS. Back Fetal Bovine Serum FBS that has been functionally tested for optimal use with all of our Tet Systems. This FBS does not contain trace levels of tetracycline or its derivatives which have been observed to interfere with proper regulation of TRE-controlled gene expression. Use of this serum ensures the maximal range of induction possible with the Tet Systems. Back Fetal bovine serum FBS approved for use with tetracycline inducible expression systems.Only Takara Bio USA functionally tests FBS to be sure that no contaminating tetracycline derivatives interfere with induced expression levels. Back Fetal bovine serum from Clontech is functionally tested to guarantee consistent results with tetracycline-inducible expression systems.Serum from other vendors that have not been functionally tested may contain tetracyclines that affect the maximum expression in Tet-Off systems, or generate background in Tet-On systems. Data here shows induction of luciferase expression from our CHO-AA8-Luc Tet-Off Control Cell Line using different sources of FBS. Back