Although employing RNAi vector systems can be slightly more involved than using synthetic RNAi reagents, the flexibility of the vector-based systems is compelling for many RNAi researchers conducting both in vitro and in vivo experiments. There are two main types of RNAi expression vector technologies on the market: short hairpin (shRNA) expression vectors and artificial microRNA (miRNA) expression vectors.

Most RNAi vectors available today employ shRNA vector technology, which typically involves shRNA expression from a Pol III promoter and may or may not employ viral delivery. These vectors express shRNA sequences, typically from a U6 or an H1 promoter, and some have inducible promoters (typically H1/TO—a tetracycline-inducible promoter).

While these vectors can be used for in vivo RNAi experiments, there are some drawbacks, including low design success rate and inability to track shRNA expression or express the shRNA in a specific target tissue.

RNAi vector delivery methods

Similar to RNAi vectors for in vitro applications, you can use either standard transfection techniques or a viral delivery method to deliver RNAi vectors in vivo.

Currently, the delivery of an RNAi expression vector in vivo without using a viral delivery system is fairly similar to delivering synthetic dsRNA in vivo. Typically, this would involve complexing the RNAi expression vector with a commonly used lipid-based in vitro transfection reagent and directly injecting into the animal. While this may be the easiest approach for delivery of RNAi vectors into animals, it has quite a few limitations, including the inability for systemic delivery and low transfection efficiencies. For these reasons, most researchers choose to use a viral delivery method when employing RNAi vectors for in vivo experiments.

Regardless of whether one chooses an shRNA or a miR RNAi vector system, the capability for viral delivery is an advantage for many in vivo approaches. Most viral delivery approaches involve either an adenoviral, retroviral (non-lentiviral), or lentiviral technology

  • Adenovirus can be used for transient RNAi expression in either dividing or non-dividing cells.
  • Retrovirus can be employed for transient or stable expression, but can only be used to transduce dividing cells.
  • Lentiviral delivery affords the most options, as it can be used for transient or stable expression in dividing or non-dividing cells; as well as neuronal cells, drug- or growth- arrested cells, or even primary cells (Table 1).

Table 1 - Lentiviral delivery offers the most flexibility for delivery of RNAi vectors to a wide variety of cells.

Viral system Transient expression Stable expression

Dividing cells Nondividing cells Dividing cells Neuronal cells Drug- or growth-
arrested cells
Contact-
inhibited cells
Adenovirus



Lentivirus
Retrovirus



HiPerform™ Lentiviral PolII miR RNAi Expression System with EmGFP

The HiPerform™ vector contains a mRNA stabilizing sequence (WPRE) and a nuclear import sequence (cPPT) which have generated up to 5-fold higher virus titers and EmGFP expression levels in many cell lines. Additionally, MultiSite technology allows you to express the EmGFP/miR RNAi cassette from CMV, EF-1a, or your own tissue specific or other in vivo appropriate promoter.

  • Achieve up to 5x higher titers (measured by GFP) allowing more cells to be transduced or higher multiplicities of infection (MOIs) to be employed Fig (X)
  • Incorporate your own tissue specific or other in vivo appropriate promoter 
  • Track miRNA expression through co-cistronic expression with EmGFP 
  • Knockdown more than one gene simultaneously through expression of multiple miRNAs from a single transcript


Figure 1A - The new BLOCK-iT™ HiPerform™ Lentiviral POL II miR RNAi Expression System with EmGFP
. The pLenti6.4/CMVor Ef-1a/V5-M5--GW/EmGFP-miR vector is driven by the CMV promotor, has the blasticidin resistance marker, and is available with co-cistronic EmGFP expression as a reporter.
 



 



Figure 1B - Higher fluorescence evident from pLenti6.4 constructs using the BLOCK-iT™ HiPerform™ Lentiviral POL II miR RNAi Expression System.
  Images taken four days following transduction of GripTite™ 293 cells at a MOI of 3 with the respective viruses.