The sequencing of the Haemophilus influenzae genome in 1995 sparked an increasing interest in functional genomics, specifically in the development of high-throughput screens that can more closely examine gene function in mammalian cells.
Unfortunately, researchers have found that conventional transfection methods have low efficiencies in mammalian cells, particularly in primary cell types.
Ravi Kane, an associate professor of chemical and biological engineering at Rensselaer Polytechnic Institute, and colleagues have developed a high-throughput technology that can determine the function of various genes within stem cells.
In their work, which appears online in the August issue of Stem Cells, the researchers constructed clonal microarrays by seeding a population of cells on micropatterned surfaces.
They then isolated the clones of interest after assaying in parallel for cellular processes and functions such as proliferation, signal transduction, and differentiation.
Kane spoke this week with CBA News about the advantages of this technology and its potential for overcoming the difficulties inherent in the transfection of primary cells.
Could give me some background on this technology?
The goal of our technology is to determine the function of various genes within stem cells. The idea is to figure out how to do this in a high-throughput fashion. If you look at the genome, it contains so much genetic information, that if you try to figure out the function of various genes one by one, it will be pretty difficult.
You need some way in which you can try to decipher the function of these various genes in a high-throughput, parallel fashion. To do that, different groups have developed different kinds of cellular microarray technologies.
The one that we have developed is a new kind of technology that has several advantages compared to those developed previously. We call our system a clonal microarray.
Essentially we start with what you could call a stem cell library. Basically, every cell in this library over-expresses a particular DNA sequence. But you have a vast collection of these. You then generate a surface which is micropatterned so that “islands” can be identified on the surface, and cells can only attach on the surface of those microscale islands.
We feed the library of stem cells onto the micropatterned surface, but we do it in such a way that almost all of the islands contain either no cells or only one cell. But since we are seeding a library, the cell in principle would be a different kind of cell. There may not be a different kind of cell on each and every island, but you may start with a library that contains over a million different kinds of stem cells, which means that each cell over-expresses a different DNA sequence.
On this island onto which the stem cell has attached, the cell can continue to divide. When it does, it generates a clonal population. So every other cell that is dividing on that particular island would be similar to the first one. It would over-express the same DNA sequence.
So on a slide the size of a microscope slide, we may have 3,500 different clonal stem cell populations. Then in parallel, we can screen for the function of these many different cell types. For example, we could ask the questions, “Does one of them grow faster than the other” or, “Is one of them more likely than other types of cells to differentiate into neurons,” or “Does one have a different shape than the others?” Really, one could ask any question that one may be interested in.
Once we identify a clone that has the property of interest, we can just isolate it from the slide, and by sequencing the DNA, we can identify the sequence whose over-expression caused that particular phenotype.
But the method is general. This particular paper had to do with developing the method and applying it to stem cells. But in reality, you could apply it to study gene function in any kind of cell.
You mentioned that it had certain advantages over existing methods. What are some of those advantages?
The advantages had to do with the nature in which one generates the stem cell library. One competing technology works by first generating a slide on which you initially spot different DNA sequences … in patterns. You then seed normal stem cells on the entire array. But depending on the DNA sequence that a particular cell is sitting on, so to speak, it could only take up that kind of DNA.
So in that case, the screening part is similar, meaning that you are in parallel screening for essentially different cell populations, because depending on where the cell is sitting, it will take up a different kind of DNA.
The only problem with this approach is that in order to generate this patterned surface, where different locations have different kinds of DNA, you have to first create and individually purify those DNA sequences. If you have 1 million kinds of DNA that you want to work with, you would have to purify these 1 million DNA sequences.
With our method, the way in which we make the library is in a sense easier, because you do not have to individually isolate each element of the library.
Is this something that you may wish to commercialize?
I think it is certainly possible. Clone pickers are commercially available that can pick up a clone of interest from a surface. And this method and the kind of micropatterned substrate that we have developed I think would fit in well with existing clone-picking technology. It could possibly even enhance the function, because we have a high-density array that allows us to create a large number of clonal populations in a small surface area. If we can use robotics to speed up the way in which we pick up clones of interest, then it could be quite interesting.
In addition, the technology used to make these micropatterned surfaces is pretty straightforward, so I think it would be easy to commercialize. I think it would be also be easy for other labs to adopt and use the technology.
Have any other labs expressed such an interest?
Not yet, because it is still pretty early; the paper has just been published.
What do you feel the next step in this research would be?
From a technological viewpoint, one thing might be to speed up the ease with which the clones of interest are isolated. Commercially available robotic systems exist that could create a powerful combination when interfaced with these micropatterned slides.
But on the fundamental research side, we now have a new tool for stem-cell research. Now the thing to do would be to apply it to fundamental studies to try to understand how different genes function in different kinds of stem cells. So I think applying the tool to learn how genetic elements control stem cells’ fate is the most promising application.