Lentiviral Gene Delivery
The XYRCOS or XYClone laser can be used as an easy alternative to single-cell micorinjection of lentiviruses into fertilized eggs.
A recent study, En masse lentiviral gene delivery to mouse fertilized eggs via laser perforation of zona pellucida, published by the National Institute of Environmental Health Sciences in Transgenici Research journal, discussed the benefits of using the XYClone laser.
The XYClone laser perforates targeted areas in the zona without micromanipulation. A novice user can utilize the system effectively to perforate the zona pellucida of free floating single cell embryos within minutes of first use. Conventional embryo microinjection requires more extensive training to develop expertise. By moving the LED light to the desired location on the zona while looking through the microscope eyepieces, the zona of large number of fertilized eggs can be perforated within minutes
By combining the easy to use laser zona perforation method and gene delivery via lentiviruses, we were able to rapidly and efficiently transduce multiple single cell embryos in a drop of KSOM media. The lentiviral vectors were pipetted directly into the culture media containing the perforated embryos allowing for en masse infection and transduction. The embryos were cultured with the virus until they developed into blastocysts and non-surgically transferred into pseudopregnant mice, so viability and gene expression could be assessed. The laser-assisted lentiviral gene delivery can be used to transduce fertilized eggs in bulk, takes less time for a novice user, and removes many of the technical limitations imposed by conventional micromanipulation and microinjection. Lentiviral gene delivery also allows for spatiotemporal control of gene expression.
The video protocol for using the XYClone laser for lentiviral delivery is also available at jove.com.