规格
Cloning Method:	Gateway®
Constitutive or Inducible System:	Inducible
Delivery Method:	Transfection
Product Line:	BLOCK-iT™,Gateway®
Product Size:	20 reactions
Promoter:	H1⁄TO
RNAi Type:	shRNA
Selection Agent (Eukaryotic):	Zeocin™
Vector Type:	BLOCK-iT RNAi Vectors

储存

The BLOCK-iT™ Inducible H1 RNAi Entry Vector Kit contains two boxes. The cloning box contains four tubes of linearized pENTR™ /H1/TO vector at 7.5 ng/tube, 250 µl 10X oligo annealing buffer, 20 µl T4 DNA ligase, 80 µl 5X DNA ligase buffer, 50 µl 5 nM lacZ2.1 control oligo, 10 µg lyophilized lacZ control plasmid, 1 ml 10 mg/ml tetracycline, 4.5 ml DNase/RNase-free water, and 2 µg each forward and reverse sequencing primer. Store the vectors, buffers, control oligo and plasmid, water, and sequencing primers at -20°C. Store the tetracycline at -20°C protected from light. The One Shot® box contains transformation reagents including twenty-one 50-µl aliquots of One Shot® TOP10 chemically competent E. coli, S.O.C. medium, and a pUC19 supercoiled control plasmid. Store at -80°C. All reagents are guaranteed stable for 6 months when properly stored.

描述

Take control with inducible RNAi-in any cell type. Take control with inducible RNAi! The BLOCK-iT™ Inducible H1 RNAi Entry Vector is used to generate stable cells that can be induced to express short hairpin RNA (shRNA). The shRNA leads to an RNAi response that results in down regulating a targeted protein. When introduced into cells expressing the tetracycline repressor (TR) protein, the induction of the RNAi pathway occurs in the presence of tetracycline. When tetracycline is not present, there is almost no change in cellular levels of protein. The BLOCK-iT™ Inducible H1 RNAi Entry Vector provides a simple, stream-lined approach to cloning shRNA. The BLOCK-iT™ Inducible H1 RNAi Entry Vector allows you to:

• Study genes whose removal results in cell death
• Control initiation of an RNAi response to match experimental needs
• Study a phenotype without activation of compensatory mechanisms that can occur when the shRNA is continually expressed

Mechanism of shRNA expression from the H1/TO RNAi cassette
An easy cloning process places a ~50-bp double-stranded DNA oligonucleotide encoding an shRNA immediately downstream of the H1/TO pol III promoter to create an H1/TO RNAi cassette (Figure 1). This H1/TO pol III promoter contains two TetO₂ sites for tetracycline-regulated expression. Cellular transcription of the dsDNA produces an shRNA molecule that is processed into short interfering RNA (siRNA) capable of inhibiting expression of the target gene. In cells that do not express the TR protein, the shRNA is constitutively expressed. When the TR protein is present, it tightly binds the TetO₂ sites within the H1/TO promoter, effectively blocking initiation of shRNA transcription. Tetracycline added to the culture medium binds to, and changes the conformation of, the TR protein. This causes the TR protein to release the TetO₂ sites, derepressing transcription (Figure 2). This results in shRNA expression and effective knockdown of the target gene (Figure 3). The BLOCK-iT™pENTR™ H1/TO entry vector has a Zeocin™ selection marker for generating clonal cell populations with integrated shRNA sequences. For your convenience, several functionally tested, blasticidin-resistant T-REx™ cell lines are available that express high levels of the TR protein. Alternatively, you can use the pcDNA6/TR or pLenti6/TR vector to generate a TR-expressing cell line in virtually any mammalian cell.

Transfer of the H1/TO RNAi Cassette
The H1/TO RNAi cassette can be easily transferred into other vectors and systems by a simple recombination reaction using Gateway® Technology. A variety of Gateway® destination vectors are available for alternative delivery and selection methods. These vectors permit viral delivery into hard-to-transfect, primary, or non-dividing mammalian cells or animals. For stable inducible RNAi analysis use a BLOCK-iT™ Lentiviral RNAi destination vector or for long-term transient inducible RNAi use the pAd/BLOCK-iT™ RNAi destination vector.

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