Frontiers Of Biology

Gene Circuits and Their Application in the Dynamic Reprogamming of Cells

Gene circuity has, in recent years, become a very important sub discipline of Biology. With far-reaching scientific and medical implications, advances in gene circuitry could be the key to milestone breakthroughs in experimentation and treatment. But at what cost would these breakthroughs come? Currently, most gene circuitry experiments are conducted by “hardwiring” a cell, or physically changing the genome of the organism. As far as medicine goes, this is a very invasive and potentially harmful treatment. Human trials for such a treatment will prove difficult, because the effects could be unpredictable and perhaps even dangerous.

Gene Circuits and Their Application in the Dynamic Reprogamming of Cells

How can we circumvent this issue? Dynamic reprogramming via gene circuitry suggests an alternative to hardwiring cells. Dynamic reprogramming would allow a scientist or doctor to alter the function of a cell via a synthetic gene circuit. Instead of physically changing the genes in the cell, the gene circuit would interact with an existing biological pathway in the cell, altering the expression of the genes involved in the pathway to change its outcome. The process is dynamic because the scientist can change the way the gene circuit acts without having to change the circuit after its implementation. Ideally, the synthetic circuit would be designed to react differently to multiple different inputs. Input 1 would cause a specific change in the biological pathway by affecting the way that the synthetic circuit affects the pathway. Input 2 would cause a different specific change in the biological pathway by a second mechanism, etc. Thus, a scientist could affect many different kinds of changes in cell function simply by adding different inputs to the gene circuit.

Dr. Katie Galloway and co-workers set out to design a gene circuit capable of altering a biological pathway that determines cell fate in a research paper entitled “Dynamically reshaping signaling networks to program cell fate via genetic controllers.” Galloway et al. designed a dual-diverter network that would interface with the pathway determining mating fate in yeast. A diverter is the kind of gene circuit used by the researchers, so called because it “diverts” the outcome of the pathway into a fate the cell would not normally assume without a specific input.

The diverters consist of RNA-based transducers, pathway regulators, and constitutive and pathway-responsive promoters. One diverter contains an RNA-based transducer which is responsive to the addition of the chemical tetracycline. When tetracycline is added, the transducer causes the over-expression of the gene Ste4, the protein product of which interacts with the pathway determining yeast mating fate such that the yeast cell assumes the mating fate. The pathway then gives positive feedback to the diverter, which continues to send the mating signal. The other diverter contains a transducer which is responsive to the chemical theophylline. When activated, the transducer over-expresses the gene Msg5, the product of which interacts with the pathway to induce the non-mating fate in yeast. The pathway then gives negative feedback to the diverter, locking the cell in the non-mating fate.

What makes this circuit dynamic is that the diverters are mutually antagonistic, meaning that, when one is active, the other must be inactive. This means there are three states: one diverter active, the other diverter active, and neither diverter active. When neither diverter is active, the pathway determining yeast mating fate functions as normal, and the cell may assume either the mating fate or non-mating fate in natural fashion. This means that the researchers could induce three different fates in the yeast cells simply by adding one input or another, or nothing at all, rather than hardwiring the yeast cell. Although this is not necessarily reprogramming of a cell, it is a step in the right direction. This experiment shows that cell fate can be affected by interfacing a synthetic gene circuit with an extant biological pathway, rather than physically altering the genes of the cell.

I had the chance to interview Dr. Galloway, and she revealed some interesting insights both about her experiment and life as a scientist. Some representative responses are listed here.

How did you become interested in signaling networks and their modification?

“I came to graduate school with the idea of building control into biological systems. I had a very particular mechanistic view of how the cell worked and wanted to figure out how to engineer it. I became interested in signaling networks in late 2006-2007 because I wanted to know more about biology and how cells integrate information to make rational decisions. In 2007, I saw a talk at the International Conference on Systems Biology that confirmed my ideas that by controlling the intercellular signaling pathways we could harness control of cell fate. With genetic circuits, I knew we could build controllers that would change the levels of gene expression. So if we could use gene expression to control the signaling pathway, then genetic circuits could control cell fate.”

What prompted you to pursue the particular issue of reprogramming with genetic controllers?

“I wanted to show that we could use gene circuits to dynamically call a program, instead of hard coding a response (by using a genetic knockout or other gene alteration). [Even] today, gene knockouts can be very time-consuming (sometimes impossible because they are lethal). So we wanted to find a less invasive scheme…thus dynamic reprogramming.”

Did you have any setbacks? How did you resolve them?

“Yes. We had setbacks, both technical challenges and administrative. Technically, integrating the two different programs was very challenging and it took a lot of work to build the entire set of plasmids we made ( > 300 plasmids). In 2009 my advisor moved from Caltech to Stanford. I remained at Caltech since my husband was at UCLA. For the remaining 4 years I worked independently and communicated with my advisor through email and skype as well as in 2-3 in-person meetings a year. This meant I had to find resources on campus (equipment, supplies, etc) to replace those that left with my advisor. It was quite challenging, but ultimately I had a lot of support both from my department and my advisor. Otherwise, I don’t know what would have happened.”

How did you know you wanted to be a scientist?

“I didn’t ‘know’ I wanted to be a scientist until I came to grad school. I was always good at science and curious about the way things worked, but I didn’t consider that I would be a scientist. I trained as a chemical engineer in college at UC Berkeley. I didn’t enjoy being in lab and the hours were long, so I figured research wasn’t for me. But then I did an internship and saw that I like research much better than industry. It wasn’t until I went to Caltech and started reading both in and out of my field that I fell in love with science. I think I needed a bigger perspective on life and how science relates…existentially. It was also this existential reasoning that kept me going through grad school in the really difficult times. That reason is thus: I came to see and believe that there is a rational mind behind the universe and that we can understand the work of that mind through science. Beyond that I saw the goodness of all that exists as well as our brief time to both enjoy exploring it and benefit others by productive work and compassion. By going back to the roots of science (e.g. natural theology), I find it much more rewarding to work in science, knowing that reason, experimentation, and meaning fit into the narrative of a world ordered by a compassionate Creator.”

What are you currently working on?

“I work as a postdoc converting skin cell into motor neurons so we can study diseases like ALS (Lou Gehrig’s disease). We are trying to figure out the molecular rules that allow us to convert cells from one type to another using transcription factors and also if we can do it more efficiently.”