Mir-XTM Inducible miRNA System (Red)

Description

The Mir-X Inducible miRNA System Red is a set of expression vectors that allows you to establish a Tet-regulated microRNA miRNA expression system in mammalian cells. An additional feature of the system is that a bright red fluorescent protein marker, mCherry, is coexpressed along with your miRNA, enabling you to verify miRNA expression in intact cells. Inducible expression of the mCherry/pre-miRNA transcript is accomplished by creating a double-stable cell line that expresses the Tet-On Advanced transactivator and that bears your customized pmRi-mCherry/miRNA vector, and then culturing the cell line in the presence of the tetracycline derivative, doxycycline. 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. 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