Traditional short hairpin RNA (shRNA) sequences are transcribed in the nucleus from a vector containing a Pol III promoter. The shRNA, containing the sense and antisense sequences from a target gene connected by a loop, is transported from the nucleus into the cytoplasm where the enzyme Dicer processes it into small/short interfering RNAs (siRNAs). At that point, it proceeds through the RNA interference pathway to target complementary mRNA and eventually knock down protein expression.

Vector design and construction

A vector expressing the shRNA is generated by designing a short, double-stranded DNA oligo encoding a sense-loop-antisense sequence to the targeted gene. On either side, this sequence will have a four-nucleotide overhang that is compatible with the entry vector construct and can be cloned into the pENTR/H1/TO or pENTR/U6 entry vector via a brief benchtop ligation reaction.

The entry vector contains the U6 Pol III-type promoter and the Pol III terminator sequence. Once the double-stranded oligo sequence is cloned into the vector, an RNAi cassette that expresses the shRNA will be created.

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Learn more about BLOCK-iT shRNA vectors

shRNA cloning into inducible and constitutive BLOCK-iT entry vectors

A vector expressing the shRNA is generated by designing a short double-stranded DNA oligo containing a sense-loop-antisense sequence against the target. The sequence has 4-nucleotide overhangs on the ends that can be ligated into the pENTR/H1/TO or pENTR/U6 entry vector via a brief benchtop reaction. The entry vector contains either the H1 promoter with two tetracycline operator sites flanking the TATA region, or the U6 Pol III type promoter and the Pol III terminator sequence. Cloning the double-stranded oligo sequence into the vector creates an RNAi cassette that expresses the shRNA.

Stable shRNA expression

Transfer your shRNA cassette into one or more of the BLOCK-iT DEST vectors to get efficient delivery and expression of shRNA in mammalian cells. Each destination (DEST) vector is designed with GatewayTechnology to easily transfer the RNAi cassette from the BLOCK-iT pENTR vector to a BLOCK-iT-DEST vector in just one hour. You choose what vector to use depending on your cell type and selection needs. Get delivery in virtually any mammalian cell type, including hard-to-transfect, primary, and even non-dividing cell types, with a choice of BLOCK-iT viral vectors:

  • pLenti6/BLOCK-iT-DEST: lentiviral vector with Blasticidin marker for fast, efficient selection for constitutive shRNA expression
  • pAd/BLOCK-iT-DEST: use for efficient, easy generation of an adenoviral vector without the need for a shuttle vector or other laborious methodologies

BLOCK-iT shRNA vectors

Vector TypeAttributesApplicationAdvantagesProducts
BLOCK-iT
Entry Vectors
Vector:
pENTR/U6
System: plasmid
Selection: none
Promotor: U6
Transient
RNAi in transfectable
mammalian
cells
-Constitutive promoter for shRNA expression
-5-minute bench top cloning of shRNA cassette
-Easy transfer of shRNA cassette to other BLOCK-iT DEST vectors
-Cost-effective, easy-to-regenerate vector
BLOCK-iT U6
RNAi Entry Vector
Kit (K494500)
Viral BLOCK-iT DEST VectorsVector:
pLenti4/BLOCK-iT 
-DEST
System: lentiviral
Selection: Zeocin
Promotor: none
Stable RNAi in virtually any mammalian cell type or animal model- Effective long-term shRNA expression from efficient lentiviral integration
- Reproducible delivery to primary and non-dividing cell types
- Easy one-hour recombination of H1/TO or U6 shRNA cassette
BLOCK-iT  
Lentiviral RNAi
Zeo Gateway
Vector Kit
(V48820)

Vector:
pLenti6 /BLOCK-iT 
-DEST
System: lentiviral
Selection: blasticidin
Promotor: none
Lentiviral- Effective long-term shRNA expression from efficient lentiviral integration
- Reproducible delivery to primary and non-dividing cell types
- Easy one-hour recombination of H1/TO or U6 shRNA cassette
- Fast selection with potent Blasticidin selection agent
BLOCK-iT 
Lentiviral RNAi Gateway
Vector Kit
(K494300)


BLOCK-iT 
Lentiviral RNAi Expression
System
(K494400)
Vector:
pAd/BLOCK-iT 
-DEST
System: adenoviral
Selection: none
Promotor: none
Adenoviral- Effective, reproducible delivery into nearly any mammalian cell type
- High titer viral stock for large numbers of experiments or animal work
- Easy one-hour recombination of H1/TO or U6 shRNA cassette without the use of shuttle vectors or other cumbersome methods
BLOCK-iT Adenoviral RNAi Expression System (K494100)
Support VectorsVector:
pLenti6/TR
System: lentiviral
Selection: blasticidin
Promotor: CMV
To generate stable cell lines that constitutively express the tetracycline repressor (TR)- Efficient delivery and long term stable integration in virtually any mammalian cell type with lentiviral delivery
- CMV promoter for high-level expression of the tetracycline repressor protein to produce very low basal shRNA expression when using the inducible system
- Fast selection of cells expressing the tetracycline repressor protein with potent Blasticidin selection agent
pLenti6/TR Vector Kit (V48020)
Vector:
pcDNA6/TR
System: plasmid
Selection: blasticidin
Promotor: CMV
Plasmid- CMV promoter for high-level expression of the tetracycline repressor protein to produce very low basal shRNA expression when using the inducible system
- Fast selection of cells expressing the tetracycline repressor protein with potent Blasticidin selection agent
pcDNA 6/TR (V1025-20)

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Resources

Questions

Send your questions to our RNAi team at RNAisupport@thermofisher.com