How do I know which reporter gene to use?
It all depends on your study, what you are trying to see, and what equipment you have available. There are a number of factors to consider when choosing a reporter. For more guidance visit our Choosing a Reporter page.
What type of equipment do I need to do in-life imaging?
The equipment you need will depend on the type of imaging you are performing.
- For nuclear imaging: a SPECT or PET machine, preferably coupled with a CT or MRI to localize the organs.
- For imaging luciferase or iRFP: an optical imager equipped with a cooled CCD camera.
- For imaging fluorescent proteins: an intravital fluorescent microscope.
- For imaging tyrosinase: a photoacoustic imager.
What is NIS? How do I image it?
NIS is the sodium iodide symporter. It is a membrane protein that drives uptake of iodine into cells. Mammals express NIS endogenously in certain tissues. Target cells and viruses can be engineered to express NIS. NIS concentrates several SPECT and PET radiotracers, and as such, cells or viruses expressing NIS can be imaged using nuclear imaging. For more information about NIS, when to use NIS imaging, and which radiotracers to use to image NIS, visit our Discover NIS page.
What is iRFP? How do I image it?
iRFP is the near-infrared fluorescent protein. Imanis offers iRFP713, which has an excitation of 690 nm and an emission of 713 nm. Unlike other fluorescent proteins, iRFP can be used for in vivo imaging. In vivo, iRFP is imaged using an optical imager with cooled CCD cameras. The imager must be equipped with an appropriate laser and filter combination. In vitro, iRFP can be imaged using a fluorescent microscope equipped with appropriate light and filter combinations. For more information about iRFP and using it for imaging, visit our iRFP page.
Why would I want to use multimodal imaging?
Different reporter genes offer different advantages for imaging studies. By combining multiple reporter genes for multimodal imaging, your study can benefit from the strengths of both imaging modalities or reporter genes. Using different reporter genes is especially helpful when considering the stages of a study from start to finish. For example, fluorescent proteins can be used in vitro during development, Fluc or NIS can be used for in vivo tracking, and iRFP or other fluorescent proteins used for ex vivo imaging to confirm results at the end of the study.
How do I know if I should use a selection gene?
Including a selection gene can help easily and quickly generate a population of high reporter gene-expressing cells. In most cases, therefore, using a selection gene is beneficial. However, selection genes are immunogenic, which can cause potential tumor rejection in immunocompetent mice. For more information about whether or not to use a selection gene, read our Science Talk blog post “Unnatural selection: using antibiotic selection genes with reporter-expressing eukaryotic cells.”
How frequently can I repeat NIS imaging in the same subject?
One of the advantages of NIS is that it is not immunogenic and can be used for long term studies; in principle, a single animal can be imaged numerous times. However, because animals have to be anesthetized for each imaging session, there may be limits on the number of images that can be obtained as a result of 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. The time needed between repeated imaging in the same subject depends on the physical and biological half-lives of the radiotracer you are using. Basically, you need to be sure that your imaging study is not contaminated by residual radiotracer signals from the preceding study. For example, pertechnetate has a half life of 6 hours so imaging can be repeated daily, while tetrafluoroborate has a half life of only 110 minutes so imaging can be repeated the same day. For more information about the half lives of different radiotracers click here.
What is replication competent virus (RCV)?
Replication competent viruses (RCV) are virus particles capable of infecting cells and replicating to produce additional infectious particles.
The existence of RCV in stable cell lines generated by lentiviral vector transduction is a safety concern. As such lentiviral vectors have been engineered to significantly reduce the likelihood of RCV production. These engineered lentiviral vectors have an excellent safety record, as there are no known reports of actual RCV production.
Although RCV remains a theoretical concern, most institutes have specific requirements related to testing transduced cells for RCV. A p24 ELISA assay is the most commonly used test to demonstrate the absence of RCV in a sample of transduced cells. Imanis offers p24 ELISA RCV testing for lentivirus; visit our Sample Analysis page for more information.
What is the source of the transgenes?
Our transgenes come from a variety of sources. Check out the table below for detailed information about the source of our transgenes.
Transgene Accession # Gene Source Description Luc2 AY738222 Photinus pyralis Codon optimized firefly luciferase with brighter signal1. hNIS U66088 Homo sapiens Human sodium iodide symporter; mediates iodide uptake2. hNISplus Mutant of
Homo sapiens Genetically modified hNIS with enhanced radiotracer uptake. mNIS AF235001 Mus musculus Murine (mouse) sodium iodide symporter; mediates iodide uptake3. rNIS U60282.1 Rattus norvegicus Rat sodium iodide symporter; mediates iodide uptake4. pigNIS NM214410 Sus scrofa Pig sodium iodide symporter; mediates iodide uptake5. RhNIS N/A* Rhesus macaque Rhesus sodium iodide symporter; mediates iodide uptake. dNIS XM_541946 Canis lupus Dog sodium iodide symporter; mediates iodide uptake6. IRES M81861
(nt 260 to 848)
EMCV Internal ribosome entry site; RNA element that mediates internal translation initiation7. eGFP AAB02572.1 Variant of GFP from Aequorea victoria Enhanced GFP; mutant of GFP with a 100-fold increase in fluorescent signal8. iRFP JN247409 Rhodopseudomonas palustris Near-infrared fluorescent protein; red-shifted fluorescent protein with an excitation/emission of 690/713 nm9. dsRed AB212907 Anopheles gambiae Red fluorescent protein variant with an excitation/emission of 558/583 nm10. DRD2 NM_016574.3 Homo Sapiens Dopamine receptor D2; endogenously expressed in the brain11. hNET NM_001172501.1 Homo Sapiens Human norepinephrine transporter; endogenously expressed by noradrenergic neurons12. SSTR2 AY236542.1 Homo Sapiens Somatostatin receptor 2; endogenously expressed 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 email@example.com for more information.
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