MISSION^® In Vivo shRNA and Lentiviral Solutions

Lentiviral products tailored to your in vivo research!

Sigma-Aldrich^® is the exclusive distributor of the RNAi Consortium (TRC) shRNA library clones in DNA or lentiviral format. Rarely, one size fits all. Your in vivo research needs are unique, so Sigma^® offers multiple configurable options that are tailored to fit your research. The available options include:

• Alternate vectors
• Titers from 10⁶ to 10⁹ TU/ml
• Multiple volumes

All custom requests will be considered—visit our Custom Services page for more details.

• Introduction to in vivo use of MISSION^® lentivirus
• Biodistribution of MISSION in vivo lentivirus
• Intratumoral injection of MISSION in vivo lentivirus
• Intramuscular injection of MISSION in vivo shRNA
• Xenograft model using MISSION lentivirus
• Selected references for in vivo lentiviral applications
• Contact us

Introduction to in vivo use of MISSION lentivirus

MISSION in vivo lentivirus is designed to address the challenges of extending an RNAi study into an in vivo system. Lentivirus-delivered shRNAs in vivo has been accomplished by either single high-titer injections, or serial lower-dose administration. MISSION in vivo lentivirus is suitable for RNAi research in animals using multiple routes of delivery.

Ready to Use

• High quality in vivo scale lentivirus suitable for single or repeat injections

No Observed Toxicity

• Animals injected with maximal viral loads displayed no effects due to VSV-G envelope or viral toxicity

Validated Use*

• In vivo efficacy of Sigma lentiviral preparations and MISSION TRC shRNAs for knockdown has been demonstrated in top tier journals

Designed for Your Needs

• Select from our highly validated MISSION TRC shRNA collection, request custom cloning, or lentiviral production of private clones

* It is important to use redundant shRNAs and proper controls to ensure results generated reflect a true biological finding, and reduce the potential for off-target effects

Biodistribution of MISSION in vivo lentivirus

MISSION lentivirus can transduce a variety of organs and tissues in vivo through multiple routes of delivery. Figure 1 shows the results of a proof-of-principle experiment in mice using a lentivirus expressing far-red fluorescent protein.

Figure 1. MISSION lentivirus can transduce a variety of tissues in vivo.* The far-red fluorescent protein (TagFP635™) was delivered via high titer lentivirus as a single bolus injection. Delivery routes tested were: intramuscular (IM)/subcutaneous (SUBQ), intraperitoneal (IP), and intravenous (IV). Values are expressed as fold change of fluorescence intensity relative to the untransduced control mouse. *Data provided by University of Notre Dame.

Intratumoral injection of MISSION in vivo lentivirus

MISSION lentivirus has been successful in direct intratumoral injections. A p55 knockout mouse was injected with 5x10⁵ LLC-1 (Mouse Lewis Lung Carcinoma) cells via a subcutaneous flank injection. The subsequent tumor was injected twice daily, for five days, with 20 µL of 1.9x10⁷ TU/ml TurboGFP™-expressing lentivirus (Product Number SHC003V). Five days after the final injection, the tumors were biopsied, stained with TO-PRO^®-3 (a nuclear stain) and imaged, see Figure 2.

Figure 2. MISSION lentivirus can be used in direct intratumoral injections.* Row A displays expression of TurboGFP™ (green) cells. The cells injected with the tGFP lentivirus show expression of tGFP, while the cells that were not injected show no tGFP expression. Row B overlays tGFP expression (green) with a nuclear stain (red). The cells injected with the tGFP lentivirus show expression of tGFP in addition to nuclear stain. The cells that were not injected show no tGFP expression, only nuclear stain. *Data courtesy of Goukassian, D., and Sasi, S.P., St Elizabeth’s Medical Center (unpublished results).

Intramuscular injection of MISSION in vivo shRNA

MISSION shRNA has been shown to knockdown expression of Smad3 in direct intramuscular injections (Carlson et al., 2008). Older muscle satellite cells have a reduced muscle regeneration capacity compared to younger muscle satellite cells due to an imbalance in endogenous Smad3 and Notch expression. The authors of this paper hypothesized that this imbalance was due to an increase in Smad3 expression in older muscle satellite cells. When they injected Smad3 shRNA intramuscularly into damaged muscle tissue, the older muscle tissue was able to recover its regenerative capacity when assayed five days post-injection. The authors found that a balance between endogenous Smad3 and active Notch controls the regenerative competence of muscle satellite cells.

Figure 3 shows hematoxylin and eosin staining of young and old muscle tissue and the corresponding Hoechst staining (blue) and myosin heavy chain antibody staining (green). BrdU staining and the eMyHC antibody staining is shown in the offset images.

Figure 3. Localized injection of MISSION in vivo Smad3 shRNA (intramuscular) rescues muscle regeneration capacity.* When Smad3 is silenced via multiple doses (50,000 TU/injection) of lentivirus carrying an shRNA to the Smad3 gene, the older muscle is able to recover its regenerative capacity (B) relative to younger muscle (A). Any properly planned in vivo experiment should always include a non-target shRNA control, SHC002V (D), and an untransduced control (C) to demonstrate the specificity of the shRNA being used. The regenerative capacity was visualized via IHC-staining for myosin heavy chain (eMyHC, green). eMyHC is an antibody that will help characterize myosin heavy chain during muscle fiber development and regeneration. *Carlson, M.E., et al., Nature 454, 528-534 (2008).

Xenograft model using MISSION lentivirus

MISSION lentivirus has been successful in a xenograft mouse model. HeLa cells were transduced with far-red fluorescent protein (TagFP635) lentivirus and injected subcutaneously into a nude mouse. Figure 4 shows strong local fluorescence of the resulting HeLa cell tumor expressing far-red fluorescent protein in mice.

Figure 4. Xenograft model using MISSION in vivo lentivirus.* In vivo fluorescence imaging of the subsequent HeLa tumor expressing far-red fluorescent protein in live mice. *Image courtesy of University of Notre Dame.

Selected references for in vivo lentiviral applications

• Wiederschain, D., et al., Single-vector inducible lentiviral RNAi system for oncology target validation. Cell Cycle 8, 498-504 (2009). Pubmed ID 19177017.
• Krishnamachary, B., et al., Noninvasive Detection of Lentiviral-Mediated Choline Kinase Targeting in a Human Breast Cancer Xenograft. Cancer Res. 69, 3464-71 (2009). Pubmed ID 19336572
• Carlson, M.E., et al., Imbalance between pSmad3 and Notch induces CDK inhibitors in old muscle stem cells. Nature 454, 528-534 (2008). Pubmed ID 18552838
• Croyle, M.A., et al., PEGylation of a vesicular stomatitis virus G pseudotyped lentivirus vector prevents inactivation in serum. J. Virol. 78, 912-921 (2004). Pubmed ID 14694122
• Pan, D., et al., Biodistribution and toxicity studies of VSVG-pseudotyped lentiviral vector after intravenous administration in mice with the observation of in vivo transduction of bone marrow. Mol. Ther. 6, 19-29 (2002). Pubmed ID 12095299

Contact Us

For questions about the library, pricing and quotes or other concerns, please email us at: RNAi@sial.com.

MISSION is a registered trademark of Sigma-Aldrich Co. LLC Label License. TO-PRO is a registered trademark of Molecular Probes, Inc. TurboGFP and TagFP635 are trademarks of Evrogen Co.