Luciferase is an excellent reporter gene for ‘quick and dirty’ studies of cell or virus fate in SCID or nude mice. Up to five mice can be imaged at one time making luciferase an excellent screening tool, and small numbers of cells can be detected, but images are two-dimensional and resolution is poor.

Importantly, researchers from the University of Minnesota recently reported that firefly luciferase is immunogenic, resulting in substantial loss in gene expression levels in immunocompetent mice over time. Hence, luciferase is not desirable for long term cell tracking studies. 

Reference: Cellular immune response against firefly luciferase after sleeping beauty-mediated gene transfer in vivo. Podetz-Pedersen KM, Vezys V, Somia NV, Russell SJ, McIvor RS. Hum Gene Ther. 2014 Nov;25(11):955-65. doi: 10.1089/hum.2014.048. Epub 2014 Sep 22. PMID:25093708

Mice: you can use optical imaging (luciferase or fluorescent proteins) or nuclear imaging (NIS). Biolumonescence imaging is very sensitive and there is no issue with background signals. Due to issues with autofluorescence from fur or rodent chow, fluorescence imaging in living animals can be challenging as it requires you to first subtract the background fluorescence signals.  If you want to obtain high resolution 3D true tomographic data, you should use nuclear reporter genes (e.g. NIS) with SPECT/CT or PET/CT imaging for best quantitative results.

Rats or larger: Due to inability of light to penetrate deep tissues and scatter, nuclear reporter genes (e.g. NIS) are the ideal genes for in-life imaging of living animals. If you need to monitor the genes long term, you will need to ensure that you use an immunogenic reporter gene (e.g. species specific NIS) to prevent host immune mediated rejection of NIS transduced cells.

A p24 titer is acquired via a p24 Gag antigen ELISA, whereas qPCR detects the amount of lentivirus DNA present in the sample.

Traditional p24 ELISA titrations measure both functional and non-functional lentivirus particles. However, it overestimates the functional titer, as the p24 protein pool includes a variable amount of free p24 and p24 associated with non-functional vector particles. The ratio can vary greatly between each lot so it is inherently inaccurate.

Following transduction, qPCR titration quantifies the number of lentivirus genome copies in a cell population as compared to a cellular reference gene. Absolute quantification via the standard curve method indicates the number of integrated genomes per cell. Thus, although qPCR titers are often lower than p24 titers, qPCR titration yields a more reliable functional titer.

Replication competent viruses (RCV) are virus particles capable of infection and production of infectious particles. Lentiviral vectors have been engineered to significantly reduce the likelihood of RCV production.

Our lentiviral transfer plasmids are self-inactivating (SIN) vectors in which the viral enhancer and promoter have been deleted. This increases biosafety by preventing mobilization of RCV and enabling regulated expression from the internal promoter without cis-acting effects of the LTR (Miyoshi et al., J Virol. 1998). This safety measure is combined with the 2^nd generation packaging system which excludes accessory viral genes necessary for efficient replication and pathogenesis in vivo. Together, these safety precautions should prevent production of RCV.

Yes! The Ovs are suspended in biocompatible media and can be injected intravenously into animals. We suggest dilution of viruses to desired dose level in sterile normal saline and kept on ice until use. 

The high titer and high purity viral stocks (listed as High Titer) are prepared specially for in vivo antitumor efficacy experiments. Many of our clients (including our research team) find these high titer stocks very appealing for studies in mice.

Recommended Imanis antibodies: by target and application

X = No antibody currently offered for this application.
¹Dohan et al. Mol Endoclinol. 2006. 20:1121-1137
²Dohan et al. J Clin Endocrinol Metab. 2001. 86:2697-270
³Tazebay et al. Nat. Med. 2000. 6:871-878

Our lentiviruses are 2^nd generation. Generation matters when considering which packaging plasmids to use to produce the lentivirus particles. Briefly, our lentiviral transfer plasmids are meant to be used in a 3 plasmid 2^nd generation packaging system:

1. Lentiviral transfer plasmid – the lentiviral plasmid encoding your transgene (ie. pLV-SFFV-hNIS)
2. Packaging plasmid - encoding Gag, Pol, Rev, and Tat. Tat is essential for 2^nd generation packaging.
3. Envelope plasmid – encoding the envelope protein (usually VSV-G) 

3^rd generation lentiviruses encode Gag/Pol and Rev on 2 separate plasmids and the transfer plasmids are Tat independent. Thus 2^nd generation transfer plasmids (Imanis products) CANNOT be packaged by 3^rd generation systems since 3^rd generation systems do not include Tat expression.

Our lentiviruses are 2^nd generation; our lentiviral transfer plasmids are meant to be used in a 3 plasmid 2^nd generation packaging system:

1. Lentiviral transfer plasmid – the lentiviral plasmid encoding your transgene (ie. pLV-SFFV-hNIS)
2. Packaging plasmid - encoding Gag, Pol, Rev, and Tat. Tat is essential for 2^nd generation packaging.
3. Envelope plasmid – encoding the envelope protein (usually VSV-G)

In vitro: a selection gene allows selection of only cells expressing your gene of interest, creating a fairly homogenous population. This is useful for determining transduction efficiency and expression. In addition, increasing the stringency of selection (by increasing the amount of selection antibiotic in the media) biases toward a population of high expressing cells.

In vivo: selection genes are immunogenic; leading to an immune response that rejects the cells or virus expressing the selection gene. Thus we recommend using selection genes only in immunocompromised mice (nude, SCID, etc).

about 10⁶ TCID50/mL). Each product will be shipped on dry ice and comes with a certificate of analysis and a protocol for amplification of new viral stocks in your lab.

• Wait 1-2 hours. 
• Place anesthetized mouse on the imaging bed. 
• Image per protocol

Recommended Imanis antibodies: by target and application

X = No antibody currently offered for this application.
¹Dohan et al. Mol Endoclinol. 2006. 20:1121-1137
²Dohan et al. J Clin Endocrinol Metab. 2001. 86:2697-270
³Tazebay et al. Nat. Med. 2000. 6:871-878

We are happy to provide the complete sequence of the plasmid transgenes to our customers upon request. Please email info@imanislife.com.

The optimal amount of lentivirus for transduction is cell-type dependent (e.g. primary cells require more virus than established cell lines). We recommend determining the optimal MOI for transduction by plating several wells of the target cells and infecting with increasing MOIs (e.g. 1, 3, 10, and 30*).

*For more information about calculating MOI, please see Cell Biology Protocols.

Our lentiviruses are 2^nd generation. Generation matters when considering which packaging plasmids to use to produce the lentivirus particles. Briefly, our lentiviral transfer plasmids are meant to be used in a 3 plasmid 2^nd generation packaging system:

1. Lentiviral transfer plasmid – the lentiviral plasmid encoding your transgene (ie. pLV-SFFV-hNIS)
2. Packaging plasmid - encoding Gag, Pol, Rev, and Tat. Tat is essential for 2^nd generation packaging.
3. Envelope plasmid – encoding the envelope protein (usually VSV-G) 

3^rd generation lentiviruses encode Gag/Pol and Rev on 2 separate plasmids and the transfer plasmids are Tat independent. Thus 2^nd generation transfer plasmids (Imanis products) CANNOT be packaged by 3^rd generation systems since 3^rd generation systems do not include Tat expression.

Our lentiviruses are 2^nd generation; our lentiviral transfer plasmids are meant to be used in a 3 plasmid 2^nd generation packaging system:

1. Lentiviral transfer plasmid – the lentiviral plasmid encoding your transgene (ie. pLV-SFFV-hNIS)
2. Packaging plasmid - encoding Gag, Pol, Rev, and Tat. Tat is essential for 2^nd generation packaging.
3. Envelope plasmid – encoding the envelope protein (usually VSV-G)

Our antibody concentrations range from 0.4 mg/mL to 1.5 mg/mL. Please refer to the COA shipped with your antibody for recommended dilutions.

Based on in-house studies, lentivirus titers drop approximately 50% with each freeze-thaw cycle.

Imanis will be able to ship the OVs to you after you have ordered them. However, most institutes require the scientist and lab to have biosafety registration and approval from the institutional biosafety review committee (IBC) prior to using and experimenting with any recombinant viruses. Please treat all oncolytic viruses as a biohazard and take the necessary actions to ensure personnel and environment protection.

For labs in the USA requesting VSV viruses, you will need to obtain a license from the United States Department of Agriculture (USDA) to certify that your lab and personnel are qualified to handle and transport VSV agents. Please refer to the USDA website for more information. NOTE: The VSV offered are derived from cDNA encoding attenuated lab adapted Indian strain of VSV and NOT wild type VSV.

Yes you can! We would love to offer your virus for research use!

Please contact us at info@imanislife.com or call us at +1-507-218-2559 (8:30 am to 5:00 pm, USA Central Time zone) to discuss further.

about 10⁶ TCID50/mL). Each product will be shipped on dry ice and comes with a certificate of analysis and a protocol for amplification of new viral stocks in your lab.

Yes! The Ovs are suspended in biocompatible media and can be injected intravenously into animals. We suggest dilution of viruses to desired dose level in sterile normal saline and kept on ice until use. 

The high titer and high purity viral stocks (listed as High Titer) are prepared specially for in vivo antitumor efficacy experiments. Many of our clients (including our research team) find these high titer stocks very appealing for studies in mice.