Discussion of a Laboratory Technique: Calcium Phosphate Precipitation on Dictyostelium discoideum

The original goals of my research included examining the physiological functions of PACSIN 2, a PCH protein family member, in zebra fish as well as tagging the protein with green fluorescent protein to examine when and where it is expressed in the developing embryos. The zebra fish has been the animal model that our lab has been using for the past couple of years. Recently my advisor added another model organism to our stocks for study, Dictyostelium discoideum. Dicty is a eukaryotic organism that has many gene analogs that are present in higher eukaryotes (dictybase.org, 2010). Thus it is of particular interest in research because research done on this organism may provide answers to questions about gene and protein function in other organisms.

My first goal in working with this new organism is to determine if the Dicty cells can use the CMV promoter to express a certain protein. The LIC vector from our stocks uses this CMV promoter and also codes for green fluorescent protein (GFP). Thus, if the Dicty cells take in the vector by transformation and express GFP then the cells must be able to use the CMV promoter.

Transformation is the main method for inducing the expression of a protein in the Dicty cells, and is a process similar to the transformation of bacterial cells. There are two common protocols used on the Dicty cells to induce uptake of the vector. Electroporation involves exposing the cells to electrical shock which induces the cells to uptake the vector. Calcium phosphate precipitation is a form of osmotic shock that causes the uptake. Following either of transformation protocols the cells are then selected for by taking advantage of a resistance gene that is also present in the vector (Gaudet, 2007).

Depending upon the intended use of the transformed cells one method may be preferable to the other. Electroporation is recommended for experiments requiring homologous recombination. The calcium phosphate precipitation is recommended for experiments that are designed to induce an over expression of a specific protein because the precipitation results in a higher uptake of vectors per cell (Gaudet, 2007). My preliminary test requires only the detection of the GFP and could be classified as an over expression experiment. Therefore I chose to use the calcium phosphate precipitation procedure.

I have never used this protocol before and am anticipating several potential issues with the experiment. The glycerol osmotic shock must be at the correct concentration or it will lyse the cells. Along with this difficulty with the glycerol it must be applied for exactly 5 minutes or the cells will be subjected to lysis. However the large population of cells used in the protocol is likely to produce viable GFP if the promoter is used by the cells. If this procedure fails due to cellular lysis I will have to repeat the experiment and possibly remake the solutions that are used in the procedure to ensure that their concentrations are correct. If repeated failure is seen with this time sensitive procedure it may be more time and cost effective to use the electroporation method despite the lower uptake of the vector that has been observed with this protocol.

Works Cited

dictybase.org. (2010, March 4th). Retrieved January 7th, 2011, from Dictyostelium discoideum: Model System in Motion: www.dictybase.org/tutorial/

Gaudet, P. P. (2007). dictybase.org. Retrieved January 7th, 2011, from Transformation of Dictyostelium discoideum with plasmid DNA : http://www.dictybase.org/techniques/transformation/gaudet_2007_transformation.html