Life Science

Genome Editing with CompoZr Custom Zinc Finger Nucleases (ZFNs)

This video demonstration will begin with an overview of the nucleofection laboratory exercise and experimental layout before moving onto the washing of cells to remove serum, preparation of ZFN reagents, nucleofection of samples, and summary of the expected exercise results.

In this exercise we will use nucelofection to transfect zinc finger nuclease reagents into K562 cells. Nucleofection is a specialized form of electroporation that enables the transfer of genetic material into a cell nucleus. This process relies on cell specific reagents and conditions.

In short, we will mix the cells and genetic material to be introduced together in a cell type specific solution supplied by Lonza. This mixture will be placed into a cuvette which is then inserted into the nucleofector. A cell type specific nucleofection protocol is selected and the nucleofector will then apply a proprietary current to the cuvette resulting in successful introduction of the genetic material into the nuclei of the cells.

This exercise has been optimized for use with K562 cells. Other cell types you may use in your own work will require their own set of reagents and conditions. K562 cells were the first human immortalized myelogenous leukemia line to be established. They have several qualities that make them useful in this exercise. In particular, these suspension cells are easily nucleofected and have a high rate of homologous recombination of nearly 30 percent.

We will be using ZFNs designed to target the human AAVS1 locus which has been established as a safe harbor site similar to the Rosa26 site in the mouse. Using this site we can insert genetic material without disrupting the function of other genes. The donor plasmid we will be using contains a multiple cloning site that will be inserted via homologous recombination into the AAVS1 site which has been cut by our ZFNs.

We will perform a total of four nucleofections, including a GFP expression plasmid only nucleofection control, ZFN mRNA only, ZFN donor plasmid only, and ZFN plus donor plasmid.

Use of these four reagents will allow us to validate the experiment. The GFP only sample will serve as a positive control for nucleofection. The ZFN mRNA only sample will allow us to verify that the ZFNs cut at the desired site and at what efficiency. The Donor plasmid only sample will allow us to test for random integration. Lastly, the ZFN mRNA and Donor plasmid sample will allow us to verify successful cutting and integration of genetic material into targeted location.