Gene expression is one of the most complex and tightly controlled processes within eukaryotic cells. Cellular fate and indeed the survival of entire organisms are crucially depend on precise spatio-temporal coordination of a multitude of genes.

Thus, in the last years there have been several approaches aiming to provide spatio-temporal control over gene expression, including  optogentic tools, which are to date the method of choice to adress these challenges. However, an obstacle of this technique is the time consuming introduction of any gene of interest under control of light inducible promoters.  (For each gene to be expressed by light induction, a  construct containing this gene under control of a light inducible promoter has first to be introduced into the cells.) This limits optogentic methods regarding the variability and number of genes to be inserted and controlled and makes the procedure time-consuming.

In contrast, a large variety of different genes can easily be packed and delivered by viral vectors. However, gene delivery with viral vectors is unspecific and lacks the ability to target specific subpopulations of cells.

For the past six months our team developed a novel application, called The AcCELLerator, which combines the advantages of both approaches. what do you use it for??? We use a blue light inducible gene expression system, which mediates spatio-temporal control and a MuLV based viral vector that provides an easy way to insert any gene cargo.

This combination allows to overcome the drawbacks of both approaches: it facilitates the delivery of variable exogenous genes and provides control over endogenous genes, both with high spatial resolution.

The principle of The AcCELLarator and how the components work together is briefly described below.

Optogenetics, a novel technology that allows temporal and spatial induction of gene expression by the use of light, is of growing importance for fundamental research and clinical applications. However, its biggest limitation is the time consuming introduction of transgenes into organisms or cell lines. In contrast, easy but unspecific gene delivery can be achieved by viral vectors. We, the iGEM Team Freiburg 2014, combine the advantages of both approaches – the temporal and spatial resolution of optogenetics, and the simplicity of gene transfer offered by viruses.

To this end we designed a system where the entry of a virus is enabled or prevented by exposing the target cells to light of distinct wavelengths.

In principle, the AcCELLerator bridges the gap between both systems by the light induced expression of a receptor that serves as the entry point for the virus. The components of the system and how they work together are briefly presented below.

Our tool is basically a two-component system, combing specificity of optogenetics with variability of viral vectors. The light system is stably integrated into a cell line. It controls the expression of a certain receptor, CAT1 (cationic amino acid transporter 1). When illuminated with blue light the light system switches into the “ON”-state and the receptor is expressed. Cells expressing the receptor can be recognized by our second component, a viral vector. The cargo of the viral vector is easily exchangeable.

With this system the delivery and stable integration of almost any gene of interest is an easy three step process: (1) Creation of viral vector which is carrying the gene of interest. (2) Specifying a subpopulation of cells via illumination with blue light. These cells will afterwards express the mCAT1 receptor. (3) Transduction of cells with the viral vector. The viral vector will only infect receptor displaying cells. By using The AcCELLerator it is easily possible to stably insert and express multiple genes with high spatiotemporal resolution in a matter of days.

Light induced expression of target genes bases on a system that consists of mainly two parts: One, a complex of LOV2 fused to Gal4DBD constantly located  at a specific DNA sequence, the Gal4UAS. While in the dark, Jα chain is not exposed, therefore, the ePDZ-VP-16 domain can not be recruited and no gene expression is detectable.

Upon illumination, the Jα chain of the LOV2-domain becomes accessible enabling the second part of the light system, epdZ fused toVP16, to bind to the Jα chain. The VP-16 domain of the second part acts as a transactivator of transcription which recruits DNA polymerase to the target gene.

Viral vectors constitute a fast and easy to use method of gene delivery. By using viral vectors it is possible to deliver and stabely express a certain gene cargo within only four days. In order to ensure safety and to enable the insertion of large gene cargos, essential viral genes are transferred to a so-called packaging cell line. This cell line can afterwards produce infectious viral particles easily, if it is transfected with a transfer vector carrying the gene of interest.

The receptor represents the key component of the AcCELLerator that is essential for the combination of viral vectors and light induced expression systems: Only, if the receptor is present on cells, the virus is able to infect them and insert the gene of interest. Otherwise, the virus can not enter the cell.

Without illumination the cells are in a dormant state and cannot be infected by the viral vector. Upon exposure to the appropriate wavelength, they start expressing the viral entry receptor CAT-1. Addition of the viral vector to the culture medium leads to infection of the activated subset of cells.

• Usage of a cell line stably expressing the light systems
• Easy insertion of genes