Traditional small/short hairpin RNA (shRNA) sequences are usually transcribed inside the cell 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 Dicer processes it into small/short interfering RNAs (siRNAs). At that point, it proceeds to knock down protein expression. Invitrogen also has BLOCK-iT™ Pol II miR RNAi Vectors, an improved vector technology that has many benefits over traditional shRNA vectors.

Invitrogen has two kits that provide a simple and streamlined approach to generate an shRNA vector. The BLOCK-iT™ U6 RNAi Entry Vectors provide constitutive expression of the shRNA while BLOCK-iT™ Inducible H1 Entry Vectors employ a promoter induced to express the shRNA sequence with Tetracycline (Figure 1). You can directly transfect mammalian cells with the BLOCK-iT™ entry vector containing your shRNA, or easily transfer your shRNA cassette to a selection of other BLOCK-iT™-DEST vectors, including vectors for adenoviral or lentiviral delivery.

Figure 1. The BLOCK-iT™ shRNA Entry Vectors

Easy shRNA cloning into inducible and constitutive BLOCK-iT™ entry vectors. A vector expressing the shRNA is generated by designing a short, doublestranded 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 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. Once the double-stranded oligo sequence is cloned into the vector, an RNAi cassette that expresses the shRNA will be created.

Get tightly regulated inducible expression

Use the BLOCK-iT™ Inducible H1 RNAi Entry Vector Kit to generate an shRNA vector in an easy, 5-minute cloning reaction. With the BLOCK-iT™ Inducible H1 RNAi Entry Vector Kit, you can directly transfect your entry clones and perform stable selection using Zeocin™. In cell lines that express the tetracycline repressor gene, you’ll have tight control over your shRNA expression so you can:

  • Control the initiation of the RNAi response to observe changes over time
  • Study phenotypic change during recovery of the target gene by ending induction of shRNA expression
  • Study loss of function in a stably transfected cell line even if the gene of interest is required for cell viability or proliferation

Or, if you just need transient shRNA expression, use the BLOCK-iT™ U6 RNAi Entry Vector Kit to introduce and express your shRNA sequence in many mammalian cell types.

More stable shRNA expression options

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 Gateway® Technology 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:

  • pLenti4/BLOCK-iT™-DEST: lentiviral vector with a Zeocin™ selection marker for compatibility with the tetracycline-regulated inducible system
  • 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

If you are using readily transfectable cell types, you can choose the pBLOCK-iT™6- DEST vector for long-term shRNA expression. Transfer the shRNA cassette into these vectors for Geneticin® or Blasticidin selection, respectively.

BLOCK-iT™ shRNA Vector Table

Vector Type Attributes    Application Advantages
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 (K4945-00)
vector:
pENTR™/H1/ TO
system: plasmid
selector: Zeocin™
promotor: H1/TO
Plasmid -Tetracycline inducible H1 promoter for low basal and powerful activation of 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™
Inducible H1 RNAi Entry Vector Kit (K4920-00)
Viral BLOCK-iT™ DEST Vectors vector:
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
- Zeocin™ selection compatible with inducible H1/TO expression in tetracycline repressor-expressing cell lines
BLOCK-iT™
Lentiviral RNAi
Zeo Gateway®
Vector Kit
(V488-20)


BLOCK-iT™
Inducible H1
Lentiviral RNAi
System
(K4925-00)
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
(K4943-00)


BLOCK-iT™
Lentiviral RNAi Expression
System
(K4944-00)
vector:
pAd/BLOCK-iT™
-DEST
system: adenoviral
selection: none
promotor: none
Adeno- viral - 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

pBLOCK-iT™
-DEST Vectors
vector:
pBLOCK-iT™6
-DEST
system: plasmid
selection: blasticidin
promotor: none
Plasmid - Long-term stable expression
- Fast selection with potent Blasticidin selection agent
pBLOCK-iT™
6-DEST (V487-20)
Support Vectors vector:
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 (V480-20)
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)