One of the first impressions experimental cell biology made on me was the time it took to do quantitative experiments. I have vivid memories of waiting weeks for radioactive gels to be exposed to film, followed by those adrenaline filled moments as the film developed in a dark room. Experiments – more often than not – took time.
Today, we routinely perform thousands of experiments a day on living cells, and image the results of systematically silencing genes on cell assays. So much has changed.
Part of the technology driving this change has been based on a change of paradigm in the way we test cells.
Enter reverse transfection
The invention of reverse transfection was one key step toward accelerating cell biology and changing the nature of cell based screening. Invented by David Sabatini and colleagues at the Whitehead institute, reverse transfection comprised the idea of printing small amounts of expression constructs with a polymer and transfection reagent onto a slide. This created a miniaturized array of individual, distinct experiments where overlaid cells were transfected on the printed spot.
My research group and others saw the potential for combining reverse transfection with the libraries of siRNA targeting each and every gene in the human genome. This effort led to us producing the first genome scaled arrayed siRNA libraries and their use to identify human host factors in HIV and Chagas disease. To make this possible required innovations from applying automated systems for imaging the arrays through to computational tools for data analysis.
One of the key steps we saw as necessary to make this technology broadly available was to develop technology to produce arrays of reverse transfection faster and at larger scale than previously possible. At the time we began applying reverse transfection, we adopted the spotting technology used for making microarrays to produce reverse transfection siRNA arrays. This was a serial, iterative process limited in scale. We spent several years R&D developing an alternative that was easier, scalable and designed for phenotypic screening from the ground up.
Like replacing the pen with a printing press
Our technology exploits the principle of a printing press to produce arrays of printed reverse transfection experiments. In place of a printing pin, we have a massive array of capillaries that simultaneously print an entire array in one contact. A good analogy is that we replaced a pen with a printing press, though one where there are thousands of different kinds of ink.
One of the challenges we face as cell biologists is that the genome is large, with more than twenty thousand genes. To be able to interrogate gene function, we had the goal to be able to handle all the siRNAs that target them and, significantly, to be able to print them all at a scale where the cost and scale enabled them to be shared. Adopting the principle of a printing press enabled us to cut the time, effort and automation steps required for producing arrays of siRNA experiments. We now have eleven issued patents on our unique printing technology.
Our printing technology was designed from the ground up to meet these challenges and produce the arrays on a new, high quality glass bottomed imaging plate. This was a key goal for us, because it delivered a miniaturized screening solution with the option for high quality imaging of many different kinds of disease or cell biological models. Each plate can hold thousands of different siRNA, essentially in a single large well. This approach makes cell culture and staining very similar to handling one individual well in a microtiter plate. Experiments for us are now arrays of spots of printed siRNA, each one optically addressable and each one silencing a unique gene. We have taken reverse transfection and scaled it. Each spot is an siRNA encapsulated with transfection reagents that deliver the siRNA into cells. Each spot is a part of a larger array where each spot functions as a separate, distinct experiment. And every spot is optically addressable, so when the array is imaged the effects of individual siRNA can be identified.
Currently, we are working on producing printed arrays featuring siRNA targeting the human Kinome. In this case, we have up to two kinomes or 1400 experiments per plate. All it takes to screen them is the time it takes to grow them with cells, fix and stain them as if they were a single well.
Five thousand experiments – a mornings work
There is a thrill to seeing thousands of experiments roll by as an automated screening imager acquires the arrays. On a typical screening day, we run close to five thousand experiments in a morning, a scale of cell biology experiments that never fails to enthrall and excite. The figure below shows an example of two spots from a validation array, 48 hours after HeLa cells were grown on them and the cells were fixed and stained. When INCENP is silenced, the slowing of cell division leads to the accumulation of spectacular, multi-nucleate cells. Experiments like these are just two of thousands taken in a day. The memories of month long experiments are consigned to the past. Gone but not forgotten.
Two spots imaged on arrays after Hela cells were overlaid on them for 48h, then immuno-stained for RELA and nuclei labelled with Hoechst 33342. Spots are shown in red.
A. Control siRNA
B. INCENP siRNA spot
Arrays were printed in Persomics Imaginearray plates and imaged on a BioTek Cytation 3 imager. Scale bar 100 µm.
The title of this blog is “going forward in reverse,” we believe that our innovation has scaled reverse transfection into a tool that enables discovery for researchers and one that helps them move forward to new applications for siRNA based screening, using reverse transfection.