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Frequently Asked Questions

Technical Questions

  • > Luciferase seems more convenient. Why and When should I use NIS?

  • 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

  • > Which reporter gene should I use for whole animal imaging?

  • 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.

  • > What is the difference between a p24 and qPCR titer?

  • 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.

  • > My biosafety office is asking me about RCV (replication competent virus). What is this?

  • 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 2nd 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.

    However, the FDA requires that lentiviral stocks used in human clinical trials be tested for RCV, and some institutions have adopted this in their biosafety protocols. A p24 Gag antigen ELISA assay is most commonly used to demonstrate that there is no increase in p24 titer over time in the media of transduced cells. Imanis offers p24 ELISA RCV testing for lentiviruses, please see this page for more information.

  • > Can the OVs be administered to animals intravenously?

  • 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.

  • > You have so many antibodies. Which one do I use?

  • Recommended Imanis antibodies: by target and application

    Target

    Western

    blotting

    Immunofluorescence

    Flow cytometry

    Immunohistochemistry

    Intact cells

    Permeabilized cells

    Intact cells

    Permeabilized cells

    Human NIS

    SJ1 (REA004)

    VJ1 (REA002)

    VJ2 (REA003)

    ETNL (REA009)

    KELE (REA010)1

    VJ1 (REA002)

    VJ2 (REA003)

    ETNL (REA009)

    Mouse a-hNIS (REA011)

    SJ1 (REA004)

    KELE (REA010)2

    Rhesus NIS

    SJ1 (REA004)

    X

    SJ1 (REA004)

    X

    SJ1 (REA004)

    X

    Rat NIS

    α-rat NIS (REA008)

    X

    α-rat NIS (REA008)

    X

    α-rat NIS (REA008)

    α-rat NIS (REA008)3

    Mouse NIS

    α-rat NIS (REA008)

    X

    α-rat NIS (REA008)

    X

    α-rat NIS (REA008)

    α-rat NIS (REA008)3

    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

  • > What generation are your lentiviruses? Why does generation matter?

  • Our lentiviruses are 2nd 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 2nd 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 2nd generation packaging.
    3. Envelope plasmid – encoding the envelope protein (usually VSV-G) 

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

  • > What packaging system should I use with your plasmids to generate lentivirus?

  • Our lentiviruses are 2nd generation; our lentiviral transfer plasmids are meant to be used in a 3 plasmid 2nd 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 2nd generation packaging.
    3. Envelope plasmid – encoding the envelope protein (usually VSV-G)

  • > How often can I repeat the NIS imaging study in the same subject?

  • This depends on the physical half life of the radiotracer you are using.  Basically, you need to be sure that your imaging study is not contaminated by residual radioactive signals from the preceding study. The SPECT tracer 99mTcO4 (pertechnetate) has a half life of 6 hours so imaging can be repeated daily.  The PET tracer B18F4 (tetrafluoroborate) has a half life of only 110 minutes so imaging can be repeated several times in a day.  If you have questions about a specific radiotracer, email to info@imanislife.com

  • > What are the advantages and disadvantages of using a selection gene?

  • 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).

    In general, we recommend using selection genes for in vitro work, but avoiding them for in vivo applications.

  • > Can I grow the OVs and propagate new stocks in my lab?

  • Yes! You can purchase the 'standard titer' stocks for this application (0.25 mL, 

    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.

  • > Are the OVs well characterized and accurately titered?

  • Yes! Each of the production runs are fully characterized by Imanis scientists and have to pass QC tests. The product will come with its certificate of analysis and are ready to use immediately in your experiments.

  • > Can NIS be used for long term studies?

  • Yes.  Unlike luciferase, which is highly immunogenic in mammals, NIS does not provoke an immune rejection response and does not affect the survival or biology of the NIS-marked cells which can therefore be studied throughout the life of the experimental subject.

  • > How many times can I NIS image a single subject?

  • In principle, a single animal can be imaged numerous times.  But because animals have to be anesthetized for each imaging session, the number of images that can be obtained is limited by IACUC rules governing exposure to anesthetic agents.  In human subjects anesthesia is not required so the number of imaging studies is limited by international radiation exposure standards.

  • > What is a typical imaging protocol to detect NIS positive cells in a mouse?

  • Imaging of a mouse previously implanted with NIS-cells or injected with NIS-vectors.
    Mouse is given 250-300 uCi of SPECT or PET isotopes intravenously (tail vein) or intraperitoneally. 
    • Wait 1-2 hours. 
    • Place anesthetized mouse on the imaging bed. 
    • Image per protocol

  • > Is NIS safe to use?

  • The gene itself is very safe to use, but keep in mind that the lentivectors containing the gene are rated Biosafety Level 2 (BSL2) and radiation training is generally required for researchers who will be conducting the 1-125 uptake assay using out NIS-IT kits. For those wanting to avoid using radiation in the laboratory, we do offer a NIS-YFP vector that allows for a non-radioactive uptake assay to analyze NIS function.

  • > How can NIS technology help me?

  • If you want to study dynamic processes in living animals, such as the migration, proliferation and death of specific cell populations, changes in gene expression, or the spread of virus infections, then NIS is most probably the ideal reporter gene to serve your needs.

  • > What is NIS?

  • NIS is a snitch. It transports radioactive iodide and other ‘NIS radiotracers’ into genetically marked, NIS-expressing cells which can then be ‘seen’ by noninvasive gamma camera, SPECT or PET imaging such that their location in the body can be accurately pinpointed.

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    Products

  • > What's the source of Imanis's transgenes?

  • Transgene

    Accession #

    Gene Source

    Description

    Luc2

    AY738222

    Photinus pyralis

    Luciferase codon optimized by Promega1.

    hNIS

    U66088

    Homo sapiens

    Transient NIS expression induces iodide uptake2.

    hNIS I147V

    Mutant of

    U66088

    Homo sapiens

    NIS genetically modified by Imanis for enhanced uptake.

    mNIS

     AF235001

    Mus musculus

    Transient expression induces iodide uptake3.

    rNIS

     U60282.1

    Rattus norvegicus

    Transient expression induces iodide uptake4.

    pigNIS

     NM214410

    Sus scrofa

    Transient expression induces iodide uptake5.

    RhNIS

    N/A*

    Rhesus macaque

    Transient expression induces iodide uptake.

    dNIS

    XM_541946

    Canis lupus

    Transient expression induces iodide uptake6.

    IRES

    M81861

    (nt 260 to 848)

    EMCV

    Independent translation of multiple proteins from a single transcript7.

    eGFP

     AAB02572.1

    Variant of GFP from Aequorea victoria

    100-fold increase in green fluorescent signal as compared to GFP8

    iRFP

    JN247409

    Rhodopseudomonas palustris

    Near-infrared fluorescent protein: excitation = 690 emission = 713 nm9.

    dsRed

     AB212907

    Anopheles gambiae

    Red fluorescent protein variant10

    DRD2

    NM_016574.3

    Homo Sapiens

    GPCR, dopamine receptor variant found in the brain11.

    hNET

    NM_001172501.1

    Homo Sapiens

    Sodium:neurotransmitter symporter family. Expressed by noradrenergic neurons12.

    SSTR2

    AY236542.1

    Homo Sapiens

    Somatostatin receptor. Highest expression in cerebrum and kidneys13.

    HSV-TK

    JQ352282.1

    HSV

    Human herpesvirus 1 thymidine kinase (UL23)14.

    hTYR

    M27160.1

    Homo sapiens

    Tyrosinase converts tyrosine to brown-pigmented melanin15

    *Sequence derived from a cDNA library. Email info@imanislife.com for more information.
    1 Promega
    2 Smanik et al. Biochem Biophys Res Commun. 1996. 226(2):339-45
    3 Pinke et al. Thyroid. 2001. 11(10):935-9
    4 Dai et al. Nature. 1996. 379(6564):458-60
    5 Selmi-Ruby et al. Endocrinology. 2003. 144(3):1074-85
    6 Uyttersprot et al. Mol Cell Endocrinol. 1997. 131(2):195-203
    7 Gurtu et al. Biochem Biophys Res Commun. 1996. 229(1):295-8
    8 Cormack et al. Gene. 1996. 173(1):33-8
    9 Filonov et al. Nat Biotechnol. 2011. 29(8):757-61
    10 Baird et al. Proc Natl Acad Sci USA. 2000. 97(22):11984-9
    11 Dearry et al. Cell Mol Neurobiol. 1991. 11(5):437-53
    12 Pacholczyk et al. Nature. 1991. 350(6316):350-4
    13 Parry et al. Mol Imaging. 2007. 6(1):56-67
    14 Koehne et al. Nat Biotechnol. 2003. 21(4):405-13
    15 Takeda et al. Biochem Biophys Res Commun. 1989. 162(3):984-90

     

  • > You have so many antibodies. Which one do I use?

  • Recommended Imanis antibodies: by target and application

    Target

    Western

    blotting

    Immunofluorescence

    Flow cytometry

    Immunohistochemistry

    Intact cells

    Permeabilized cells

    Intact cells

    Permeabilized cells

    Human NIS

    SJ1 (REA004)

    VJ1 (REA002)

    VJ2 (REA003)

    ETNL (REA009)

    KELE (REA010)1

    VJ1 (REA002)

    VJ2 (REA003)

    ETNL (REA009)

    Mouse a-hNIS (REA011)

    SJ1 (REA004)

    KELE (REA010)2

    Rhesus NIS

    SJ1 (REA004)

    X

    SJ1 (REA004)

    X

    SJ1 (REA004)

    X

    Rat NIS

    α-rat NIS (REA008)

    X

    α-rat NIS (REA008)

    X

    α-rat NIS (REA008)

    α-rat NIS (REA008)3

    Mouse NIS

    α-rat NIS (REA008)

    X

    α-rat NIS (REA008)

    X

    α-rat NIS (REA008)

    α-rat NIS (REA008)3

    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

  • > Can I get the complete sequence of the plasmid transgenes?

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

  • > How much of your lentivirus should I use for a transduction?

  • 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.

  • > Are the OVs well characterized and accurately titered?

  • Yes! Each of the production runs are fully characterized by Imanis scientists and have to pass QC tests. The product will come with its certificate of analysis and are ready to use immediately in your experiments.

  • > What generation are your lentiviruses? Why does generation matter?

  • Our lentiviruses are 2nd 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 2nd 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 2nd generation packaging.
    3. Envelope plasmid – encoding the envelope protein (usually VSV-G) 

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

  • > What packaging system should I use with your plasmids to generate lentivirus?

  • Our lentiviruses are 2nd generation; our lentiviral transfer plasmids are meant to be used in a 3 plasmid 2nd 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 2nd generation packaging.
    3. Envelope plasmid – encoding the envelope protein (usually VSV-G)

  • > What if I need an OV or lentivector not in the product list?

  • We can custom design and make it for you! Please contact Imanis 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 your research needs.

  • > What is the concentration of your antibodies?

  • 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.

  • > How much does the lentivirus titer drop with freeze-thaw cycles?

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

  • > Do I need biosafety approval to receive OVs?

  • 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.

  • > Can I add my viruses to the repository?

  • 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.

  • > Can I grow the OVs and propagate new stocks in my lab?

  • Yes! You can purchase the 'standard titer' stocks for this application (0.25 mL, 

    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.

  • > Can the OVs be administered to animals intravenously?

  • 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.

  • > Which viruses are offered?

  • Imanis has exclusive rights to offer replication competent recombinant measles viruses and vesicular stomatitis viruses. Many of these viruses encode reporter genes, including NIS, to meet for your research needs. Vaccinia and herpes simplex viruses are coming soon!

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    Services

  • > What are the acceptable formats for the image analysis files?

  • We accept most data files from the imaging machines. DICOM and NIFITI files are the most common files that are received by our analysts. If necessary, data files can be re-loaded and save into the preferred files formats.

  • > I need help with analyses of my image files!

  • We can help! Please email Imanis to enquire. We will first have a consultation with you to determine your needs, review your data files to determine the best course of action to get the most out of your data. We can provide ROI (region of interest) analysis, provide you with presentation or publication ready images and movies. Let's talk!

    Shipping

  • > I live outside the USA. Do I need an import license to receive the viral products?

  • Each country has its own set of rules and regulation regarding import of infectious agents. Shipping will be on dry ice via Fedex courier service. A valid import permit might be needed before the product can be shipped to the designated country. We will work with you to ensure safe and timely delivery of the products ordered. Do not worry! We have successfully shipped our products internationally and within the United States.

  • > Do I need biosafety approval to receive OVs?

  • 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.

    Permits

  • > Do I need biosafety approval to use your lentivirus?

  • Check with your institution’s biosafety officer to confirm the requirements of lentivirus use.

    Generally, lentiviral work involving animals must have both IBC and IACUC approval, and in vitro lentiviral application should be performed under BSL-2 or BSL-2+ containment practices.

  • > My biosafety office is asking me about RCV (replication competent virus). What is this?

  • 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 2nd 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.

    However, the FDA requires that lentiviral stocks used in human clinical trials be tested for RCV, and some institutions have adopted this in their biosafety protocols. A p24 Gag antigen ELISA assay is most commonly used to demonstrate that there is no increase in p24 titer over time in the media of transduced cells. Imanis offers p24 ELISA RCV testing for lentiviruses, please see this page for more information.

  • > I live outside the USA. Do I need an import license to receive the viral products?

  • Each country has its own set of rules and regulation regarding import of infectious agents. Shipping will be on dry ice via Fedex courier service. A valid import permit might be needed before the product can be shipped to the designated country. We will work with you to ensure safe and timely delivery of the products ordered. Do not worry! We have successfully shipped our products internationally and within the United States.

  • > Do I need biosafety approval to receive OVs?

  • 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.

    Distribution/Licensing

  • > Can I add my viruses to the repository?

  • 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.

    Use Fewer Animals

    Our Reduction Campaign

    At Imanis, we are committed to promoting the practice of the 3Rs in animal research. Learn how we are decreasing the use of animals and research as well as saving up to 15% on your orders. Continue reading...

    Use Fewer Animals

    Our Reduction Campaign

    At Imanis, we are committed to promoting the practice of the 3Rs in animal research. Learn how we are decreasing the use of animals and research as well as saving up to 15% on your orders. Continue reading...