Imanis Life Sciences


Science Talk

Radiotracers for NIS imaging studies March 03 2017

Choosing the best radiotracer for NIS imaging studies

Imanis Life Sciences | Rochester MN 

There are a lot of reasons to love the nuclear reporter NIS: it is non-immunogenic, gives beautiful high resolution 3D images, works for deep tissue and large animal imaging, is fully quantitative, and is translatable to the clinic. It’s an impressive list; maybe impressive enough that you want to try NIS imaging. But now what do you do? How do you get started?

Well, NIS concentrates certain radiotracers in cells, which can be imaged by PET or SPECT imaging. So first you need to decide what radiotracer and modality to use. We know this can seem intimidating, especially if you’ve never done nuclear imaging before. So let's review the most commonly used tracers and give a little more information about each to help you make the best decision for your study.

Common PET radiotracers for NIS:

18F-BF4 (tetrafluoroborate) is the prototype radiotracer. It’s relatively easy to synthesize, has high yield emission, minimal background signal, and a low absorbed radiation dose. All of these qualities make 18F-BF4 the most widely used radiotracer. However, it ranks squarely in the middle for NIS affinity, meaning that you will need more of the tracer to get a good signal. And because of its short half-life, 18F-BF4 must be synthesized and used immediately for imaging, which can make scheduling your study more difficult.

124I has a relatively long half-life for a PET tracer, which makes it a convenient choice; you can purchase 124I in advance and then you get a whole day to complete your imaging. However, it has lower yield emission, a higher absorbed radiation dose, and lower sensitivity when compared to 18F-BF4.

A recent paper by Khosnevisan et al. in The Journal of Nuclear Medicine suggested that a new fluorine radiotracer is on the horizon for PET imaging. In their study, Khoshnevisan et al. demonstrated that 18F-SO3F has a higher affinity for NIS than the existing radiotracers and minimal background signal in vitro and in vivo. We are looking forward to seeing more of this substrate in action.

Common SPECT radiotracers for NIS:

99mTc-pertechnetate is the most commonly used radiotracer for SPECT imaging of NIS. It’s clinically approved, relatively affordable, readily available, and has minimal background signal in a variety of tissues. And compared to the other SPECT radiotracers, it has the highest affinity for NIS. 99mTc-pertechnetate also has a short half-life that facilitates rapid data collection and limits radiation exposure, which is important for clinical applications.

123I is widely used for diagnosing and monitoring thyroid diseases in the clinic. However, 123I is a less popular choice for imaging studies because it has a lower affinity for NIS than 99mTc, is relatively expensive, and is not widely available.

125I has a relatively long half-life (60 days). This means that like 124I, you can purchase 125I in advance and have more time to acquire images on the day of imaging. Like 123I, 125I is clinically translatable, and is in fact a core component of several clinically-approved radiopharmaceuticals. However, 125I has a lower affinity for NIS than 99mTc. It also has a lower energy, which leads to lower tissue penetration, so it’s not suited for imaging anything bigger than mice.

Radiotracers for NIS imaging

Choosing a Modality

So PET versus SPECT, how do you choose? Both PET and SPECT are useful for quantitatively detecting deep tissue signal. Both are directly translatable to the clinic. And both are highly sensitive as compared to other imaging modalities. In general, PET sensitivity is superior to SPECT. However, PET radiotracers have shorter half-lives and must be injected quickly after synthesis. Because of this, in-house synthesis of PET radiotracers is usually necessary, which adds to the overall cost and complexity of PET imaging. While SPECT imaging is traditionally more cost-effective and convenient, it tends to be about 10 times less sensitive than PET imaging.

But when choosing a modality, one of the most important factors is probably what you have available at your organization. Imaging equipment is expensive. New models are often superior to old models – so, for example, a new SPECT machine may actually offer better resolution than an old PET one. Plus, personnel must be certified to work with CT (x-rays) and be trained to operate the equipment. Because of these practical considerations, it’s important to use what is already available to you.

So with these considerations in mind, it’s time for you to start NIS imaging. After all, with the kind of information you can get from a NIS study, it’s certainly worth giving it a try.

Carolyn (Botting) Bomidi, M.S.
Senior Research Technologist at Imanis Life Sciences 
Carolyn photo
Rianna Vandergaast, Ph.D.
Scientist at Imanis Life Sciences

Science Talk: which antibiotic selection gene? February 08 2017

Unnatural selection: using antibiotic selection genes with reporter-expressing eukaryotic cells

Imanis Life Sciences | Rochester MN 

I grow a lot of reporter cell lines, and most of them are under antibiotic selection. Sure, it takes me more time to prepare culture media, but the tradeoff is that I have populations of high reporter-expressing cells that were a lot less work to make than if I had done clonal selection.

When to use (or not) selection

So how do you know if you should include a selection gene when making a new reporter cell line? Well, the main advantage of selection genes is that you can relatively quickly weed out cells that aren’t expressing your reporter. Without a selection gene, you are forced to be content with a mixed population of cells – that may include a lot of non-reporter cells – or to perform time consuming clonal selection and screening. In general, having a selection gene offers a distinct advantage. And even if you don’t end up using antibiotic selection, having the selection gene in your cells usually won’t impact your study.

Of course, usually is the key word. Selection genes are immunogenic. This means that if you want to put your cells in an animal, having the selection gene may cause the cells to be rejected – see my previous post about immunogenicity for more information about potential tumor rejection. Now, if you’re already using an immunogenic reporter, adding an immunogenic selection gene doesn’t change the game. But if you’re using a nonimmunogenic reporter (like NIS) adding a selection gene can make a big difference. So depending on the type of in vivo study you have planned, you may want to skip the selection gene.

What selection gene to choose

Okay, you’ve decided to use a selection gene, now how do you choose one? After all there are about half a dozen antibiotics routinely used for eukaryotic cell selection (see Table 1). Well, my personal favorite is the puromycin resistance gene. Selection with puromycin is rapid (3-6 days) and the required doses are quite small (1-10 μg/mL), which make it fast and effective. G418 is a popular choice; it works well, but requires longer selection times (10-14 days) and quite high concentrations (up to 2 mg/mL) for many cell lines. The other antibiotics and their corresponding selection genes also work well. Really, when deciding, a lot comes down to convenience. Maybe your lab already has vectors for one of the selection genes or has worked out G418 kill curves for all of the cells you use. Basically, it’s hard to go wrong when choosing an antibiotic. 

Common antibiotics for eukaryotic cell selection

Tips for using selection genes

So we’ve covered when to use selection and what gene(s) to choose. Since who is you and where is your lab, that just leaves us with how to use selection. I’m not going to go into specific protocols, but I will mention a few tips and considerations to help you get the best results.

When using selection, start with a kill curve. Doing the kill curve will help you establish a baseline for how much antibiotic is required to kill cells that aren’t expressing the selection gene. And remember, the amount of antibiotic needed varies between cell lines, so you will want to repeat the kill curve for each new parental cell line you use. To do a kill curve, incubate parental cells with increasing concentrations of your antibiotic (see Table 1 for an starting point of what ranges to try) and observe for cell death. If using an antibiotic that takes longer to kill cells, you may need to pass your cells a few times before the kill curve is done.

I like to think of selection as having two parts: selection and maintenance. For reporter cell lines with selection genes, I recommend maintaining the cells in antibiotic at a concentration that kills greater than 95% of parental cells. By maintaining the cells under selection, you ensure that the cell population continues to consist of high reporter-expressing cells. Sure, a few – and in many cases a lot of – passages without antibiotic aren’t likely to spell disaster, but hey, why take the risk. And if you’re planning to implant the cells into animals, there’s no reason to remove the antibiotic from your culture medium. Just wash and resuspend the cells in phosphate buffered saline prior to implantation. When you do want to remove the antibiotic is when preparing freezing medium or initially thawing any cells.

So that covers the maintenance part, what about the selection part? Well, for selection I recommend using a higher concentration of antibiotic. To determine an appropriate concentration you can either guess and hope that your cells don’t all die – if you guess right you just saved some time – or you can perform a kill curve on your transfected/transduced cells. Once you’ve settled on a selection concentration, grow your cells for a few passages at the higher concentration to efficiently kill any parental cells, then move them to the lower maintenance concentration. Now for those of you working with suspension cells, it can get difficult to separate dead cells out of your population during selection. For that reason, I always do clonal selection or FACs on suspension cells following antibiotic selection. And since I’ve already done antibiotic selection, I can get away with screening a lot less clones.

One last thing to keep in mind is whether you want to make double (or triple) reporter cell lines. If you are adding multiple reporters and they aren’t linked on a single construct, you will want to use a different selection gene with each reporter gene. Then make sure to select and maintain the cells in media with both antibiotics so that you are selecting only for cells that express both of your reporters.

So there you have it. Antibiotic selection is a wonderful thing. It can save you time and more importantly improve the quality of your reporter cell populations. 

Rianna Vandergaast, Ph.D.
Scientist at Imanis Life Sciences

Science Talk: problems with immunogenic reporter genes January 25 2017

Betting against immunology: Choosing the right reporter gene for your oncology models

Imanis Life Sciences | Rochester MN 

It seems like there’s a lot of confusion about the immunogenicity of tumor models expressing reporter genes. In fact, the question I may get asked the most here at Imanis is probably whether our murine cell lines will grow in syngeneic immunocompetent mice. It doesn’t seem like a hard question: yes or no. But in fact, the answer isn’t all that straightforward, which is probably why there is so much confusion about this topic. 

When it comes to growing tumor models in mice, the basic principle is pretty simple: self is good, non-self is bad. So if you want to grow tumors in Balb/c mice, for example, you’d better pick a tumor model initially isolated from Balb/c mice. Of course, there are plenty of tumor models – like all those human ones – that don’t have syngenic mice strains. That’s when immunocompromised mice come in. Because immunocompromised mice lack or have limited immune systems, they don’t reject implanted cells and tumors can grow. Not too confusing yet, right?

Okay. Now, for those of us wanting to use reporter cell lines, there’s an additional factor we have to consider: the reporter itself. A firefly is not a mouse. When our cells express firefly luciferase, we can track them noninvasively in vivo, but the trade off is that the cells are now expressing a non-self protein. And that means that the cells are now immunogenic and might be rejected by immunocompetent mice. So the answer to the question of whether our cells will grow in immunocompetent mice must be no, right? Well, maybe.

There are numerous examples in the literature of people growing luciferase cell lines in immunocompetent mice. And luciferase-expressing cells can form tumors and metastases in immunocompetent mice, in fact they often do so very well. Great, it works! Let’s throw immunology out the window.

Not so fast. The problem is consistency. Some mice do reject the cells and there’s no good way to predict when or how often this rejection will occur – though we have noticed it seems to be a little more common with some models compared to others. And the longer your study goes, the more opportunity for the immune system to mount a response against the immunogenic reporter.

So that’s why I always recommend that cells expressing any immunogenic reporter – luciferase, fluorescent proteins, near-infrared fluorescent protein (iRFP), etc. – or selection genes – puromycin, neomycin, etc. – be used in immunocompromised mice. It’s not that I think the cells are definitely going to get rejected in immunocompetent mice, it’s that I can’t promise consistent results.

So what do you do if you need to use an immunocompetent model? Fortunately, there is a murine reporter available: the murine sodium iodide symporter (or mouse NIS). Because NIS is a self-protein, it doesn’t elicit an immune response in immunocompetent mice. It’s just one of the reasons we love NIS. And for anyone working with other animals, NIS comes in a variety of species specific “flavors”.

But maybe you don’t want to or can’t switch to NIS imaging. Well, then I guess the question you really need to ask yourself is: can I afford to gamble?

Rianna Vandergaast, Ph.D.
Scientist at Imanis Life Sciences
Choosing right reporter gene for cell tracking imaging
Interesting Reads:
1. The enhanced GFP is minimally immunogenic in C57B6 mice by Skelton et al. Gene Therapy. 2001, 8(23): 1813-1814.
2. Cellular immune response against firefly luciferase after sleeping beauty-mediated gene transfer in vivo by Podetz-Pedersen et al. Hum Gene Ther. 2014, 25(11):955-65. 

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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...

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