Short hairpin RNAs (shRNAs) are a vector-based solution used to silence gene expression via RNA interference (RNAi) pathways. Our shRNA vectors are designed to deliver robust and stable gene silencing through the expression of shRNAs, which are subsequently processed into small interfering RNAs (siRNAs) within the cell, leading to targeted mRNA degradation and eventually suppressing protein expression. We have a diverse range of shRNA vectors, each optimized for efficient and reliable knockdown of specific genes. Whether your experiment requires constitutive or inducible expression, our vectors are equipped with various promoters and selection markers to suit experimental needs.

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shRNA 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 shRNA 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 sequence will be created.

shRNA cloning into inducible and constitutive 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 shRNA 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 into the vector creates an RNAi cassette that expresses the shRNA sequence.

Stable shRNA expression

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

  • 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

shRNA vectors: Selection guide

Choose a shRNA vector based on applications and advantages.

Vector typeAttributesApplicationAdvantagesProducts
BLOCK-iT Entry VectorsVector:
pENTR/U6
System: plasmid
Selection: none
Promoter: 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
U6 RNAi Entry Vector Kit
Viral BLOCK-iT DEST VectorsVector:
pLenti4/BLOCK-iT-DEST
System: lentiviral
Selection: zeocin
Promoter: 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
Lentiviral RNAi Zeo Gateway Vector Kit
Vector:
pLenti6/BLOCK-iT-DEST
System: lentiviral
Selection: blasticidin
Promoter: 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
Lentiviral RNAi Gateway Vector Kit

Lentiviral RNAi Expression System
Vector:
pAd/BLOCK-iT-DEST
System: adenoviral
Selection: none
Promoter: 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
Adenoviral RNAi Expression System
Support vectorsVector:
pLenti6/TR
System: lentiviral
Selection: blasticidin
Promoter: 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
Vector:
pcDNA6/TR
System: plasmid
Selection: blasticidin
Promoter: 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

Order shRNA vectors

shRNA vectors
Resources
Questions?

Technical inquires:
Our Technical Application Scientists are available to help assist you at techsupport@thermofisher.com

Ordering & Order Status inquires:
If you have questions about pre-designed RNAi orders and order status, please contact us at genomicorders@thermofisher.com

If you have any questions about Custom RNAi orders and order status, please contact us at RNAiSupport@thermofisher.com

For Research Use Only. Not for use in diagnostic procedures.

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