shRNA

Advanced RNAi Applications — in vivo FAQs

Can our lentivirus be utilized in vivo?
Is it beneficial to exchange Sigma® buffer for PBS?
Where does the virus go when I inject it?
How should I track my in vivo experiments?
Can I deliver plasmid DNA in vivo?
What controls do I need for an in vivo experiment?
What are the pros/cons of in vivo vs. ex vivo delivery?
How long does shRNA-mediated knockdown last in vivo?
Are there any toxic effects of shRNA in animal models?
Which tissues have researchers direclty delivered shRNA into?
Do you have a specific in vivo-grade lentivirus, or diluent to suggest?
Does lentivirus exhibit the same toxicity as adenovirus in vivo?
For additional information on in vivo applications, please visit our MISSION® In Vivo and Lentiviral Solutions page.


Can MISSION lentivirus be utilized in vivo?
Yes! MISSION produced lentivirus has been demonstrated to function in vivo as published in Nature.1 In addition, through our collaborators, the MISSION lentivirus has been successfully delivered via a variety of methodologies including: Intravenus (IV), Intraperitonial (IP), Intramuscular (IM), Intratumoral, ex-vivo.

Is it beneficial to exchange Sigma buffer for PBS?
Sigma can offer high-titer virus in various diluents upon customer request. There has been some feedback that virus suspended in PBS may lose some of its functionality relative to our traditional diluent (DMEM + 10% serum). However, we haven’t received data in support of or in contrast to the claim of reduced stability in 1x PBS.

Where does the virus go when I inject it?
This is an area of research Sigma is diligently pursuing. Preliminary results suggest that the virus is able to transduce a variety of organs/tissues when delivered systemically, including: muscle, liver, kidneys and lung.

How should I track my in vivo experiments?
The simplest method for tracking in vivo experiments is via fluorescence. We offer a variety of fluorophores that can be knocked into your cells via our lentivirus delivery system. Conversely, our custom shRNA vectors, can be concurrently expressed with a reporter fluorophore. Because the fluorophore is driven by a pol II promoter, if a cell has been transduced, and that promoter is active in that tissue, then fluorescence can be observed. A negative result for fluorescence does not definitively demonstrate a lack of transduction.

Can I deliver plasmid DNA in vivo?
Yes, plasmid DNA can be delivered in vivo. However, with plasmid delivery, the vast majority of cells will only be transiently transfected with the plasmid and silencing will only be transient. For transient gene silencing, In Vivo Quality and iScale Oligos™ siRNA would be a superior solution. That said, review the following publication for details of one method to deliver plasmid DNA shRNAs in vivo.2

What controls do I need for an in vivo experiment?
The empty vector (Product No. SHC001V or SHC001H) and (Product No. SHC002V or SHC002H) are critical for demonstrating that your new in vivo phenotype isn't due to either: delivery methodology and viral integration (Product No. SHC001V or SHC001H), engagement of the RNAi machinery (Product No. SHC002V or SHC002H), or a potential immune response (Product No. SHC001V or SHC001H), however unlikely. In addition, in order to verify your phenotype it is often necessary to silence with an independent shRNA clone or recapitulate the phenotype via a small molecular inhibitor or independent siRNA.

What are the pros/cons of in vivo vs. ex vivo delivery?
There are benefits of both delivery routes. Depending on your application and research goals, one may be better than another. Delivery ex vivo allows for complete selection of a pure population of transduced cells, and subsequent transplantation. However, direct injection of lentivirus into a discrete area may be more called for. Either option can be achieved with Sigma lentivirus.

How long does shRNA-mediated knockdown last in vivo?
There isn't an expected difference in knockdown duration between in vitro and in vivo studies.

Are there any toxic effects of shRNA in animal models?
The toxic effect can be due to introduction of off-target effects which could be sequence-dependant when the shRNA sequence has homology to mRNA transcripts other than the gene of interest.

Sequence-independent off-target effect can trigger a stress related interferon response. While the induction of interferon-pathway during RNAi is usually attributed to long, double-stranded nucleic acids, it can also be a concern during transduction of shRNA molecules. Stress responses may affect general cellular expression levels and can confuse and mislead the interpretation of data obtained from RNAi experiments.

  • Innate immunity mechanisms (IFN) can be triggered by expressed shRNA.
  • Critical cellular factors (i.e. exportin 5) can be saturated.
  • Genes other than the intended target can be silenced.

Which tissues have researchers direclty delivered shRNA into?
Systemic, brain, airways, lung, liver, mammary glands, peritoneum, muscle and joints.

Do you have a specific in vivo-grade lentivirus, or diluent to suggest?
The research community has found MISSION lentivirus to be an effective means for in vivo experimentation. Some projects require high-titer virus, in the area of 1e9 TU/mL, which we can accommodate. Other experiments may require larger volumes of the virus, again, we can easily accommodate this request as well.

Regarding a diluent suggestion, feedback that we have gotten from researchers is that if you are going into mice, the standard media formulation (DMEM + 10% serum) should work appropriately. It is when the researcher is going into higher order animals (i.e. rats, rabbits) that the immune response triggered by the serum becomes an issue. In this case, PBS may be a better alternative. But, please keep in mind that this isn’t necessarily a black and white issue; each researcher should take their experimental conditions into consideration before deciding which would better suit their needs.

Does lentivirus exhibit the same toxicity as adenovirus in vivo?
It is known for fact that lentiviruses transduce cells with the higher efficiency and toxicity effect is lowered by presence of VSV-G-pseudotyped envelope. However, toxicity will vary on a case-by-case basis.

Advantage of Lentiviral vectors is that they appear to be able to mediate persistent in vivo expression in a host of different cell types. However, the duration of expression in the context of a human clinical trial has not yet been determined. The disadvantages of adenoviral vectors is that they can mediate robust expression in some cells and that expression is transient, due in part to the inflammation and cell-mediated immune response directed against the transduced cells.

References

  1. Carlson, M.E. et al. Imbalance between pSmad3 and Notch induces CDK inhibitors in old muscle stem cells. Nature 454, 528-532 (2008). Pubmed ID 18552838
  2. Stein, U. et al. Complete In Vivo Reversal of the Multidrug Resistance Phenotype by Jet-injection of Anti-MDR1 Short Hairpin RNA-encoding Plasmid DNA. American Society of Gene Therapy. 16:1, 178–186 (Jan. 2008).

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