Our genetic blueprint consists of thousands of genes (more than 30,000) with new genes being discovered and added to the growing list. Our genes provide DNA instructions to the protein-making machinery in our bodies. These instructions can influence our health and dictate if we will get debilitating diseases. Have you ever wondered how scientists unlock which genes are responsible for what? For example, does gene A control our hair colour or gene B dictates if we will develop an autoimmune disease such as multiple sclerosis? The answer lies in DNA recombination technology which allows scientists to delete, invert or replace DNA instructions. The technology called Cre-lox recombination relies on the use of an enzyme called Cre recombinase which can bind, cut and recombine DNA at specific sites that are inserted in pairs in the DNA. The Cre-binding site in DNA is called the LoxP sequence that consists of 34 nucleotides DNA sequence made up of two inverted repeats separated by a spacer. Cre enzymes can recognize these LoxP sequences and edit the stretch of DNA resulting in gene deletion or inversion.
In a recent research article, Dr. Olaya Fernandez Gayol and colleagues use an advanced version of Cre-lox technology called DIO (Double Floxed Inverted Open reading frame) to understand the role of the Interleukin-6 (IL-6) gene in multiple sclerosis (MS). MS is a chronic disease of the brain and spinal cord in which our immune system eats away the myelin sheath around nerves disrupting the communication between the brain and the body. IL-6 is a proinflammatory cytokine known to promote MS. Gayol et al use an experimental mouse model of MS which acutely develops brain inflammation called encephalitis (Encephalo- “the brain” + itis “inflammation”) within 3 weeks of disease induction. This mouse is referred to as EAE (Experimental Acute Encephalomyelitis) which closely mimics human MS disease.
Scientists have conventionally studied the role of IL6 in EAE mice by irreversibly deleting the IL6 gene in one cell type. However, the results were confounding due to the compensatory expression of IL6 from other cell types. Gayol et al circumvent this problem by wiping out IL6 from all the cells and then recover IL-6 expression specifically in the microglial cells. It is akin to entering a dark room and turning ON a light switch at one corner of the room to clearly see what’s lying there.
Figure 1. Cartoon depicting the genetic strategy used by Goyal et al to recover IL6 gene expression exclusively in microglial cells in the mouse brain. Created with Biorender.com.
Olaya and the team use the cutting edge DIO method to wipe out IL6 and introduce the inverted form of the IL6 gene which makes this gene non-functional (Figure 1A). This inverted form of the IL6 gene does not produce IL-6 protein and mice carrying the inverted IL-6 gene (referred to as IL6-DIO-KO) are healthy (Figure 1A). As shown in figure 1B, Cre mediated recombination flips the IL6 gene in the correct orientation to make it active. The IL6 gene flipping occurs exclusively in the microglial cells and only upon treatment of mice with tamoxifen (TAM) drug. Mice in which IL-6 expression is active (referred to as IL6-DIO-ON) develop EAE disease (Figure 1B).
The team carefully optimized the duration of tamoxifen treatment in mice. Just 5 days of TAM did not flip the IL6 gene, so they extended the drug treatment to 11 days and found the IL6 gene turned on in all IL6-DIO-ON mice. Olaya says it is important to validate when creating new mouse models. “We used EAE to validate the mouse because it was a model readily available in our lab and IL6KO [deficient] mice happen to be completely resistant to the disease.” Their interesting finding that IL6-DIO-ON with IL6 gene active exclusively in microglia indicate that IL6 made in the brain promotes disease in the EAE mouse model.
As compared to more traditional methods of generating gene mutation which requires extensive mice breedings or continuous drug treatment, the strategy presented by Olaya and colleagues is labour and cost-effective. Their findings showed that in the absence of IL-6, EAE disease does not develop in mice. On the other hand, turning on the IL-6 gene (like a gene-switch) using DIO technology, mice develop the disease. Overall, this technology is highly customizable to understand the role of different genes in specific cell types in the disease context. It paves the way to gain a deeper insight and more thorough analysis of different molecular blocks involved in disease.
Dr. Olaya Fernandez Gayol is a postdoctoral research scientist in the Department of Pediatrics and co-president of Columbia University Postdoc Society(CUPS). She also manages the CUPS Press office that provides postdocs with a platform to publicize their science while improving their science communication skills.