Takara Mir-X Inducible miRNA System (Red) \Each

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.

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.

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

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.

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.

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.

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.

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.

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.