Development of a Microinjection Platform for Chlamydomonas Reinhardtii

The ability of the microalga Chlamydomonas reinhardti to produce high-value chemicals in a mild environment makes this species conducive towards low-environmental impact production practices [1-2]. Genetic manipulation of the unicellular organism to produce such desired molecules has been practiced since the late 1980s [3]. While there currently exist several functional transformation methods, first generation expression rates remain low, peaking around 60% in laboratory environments [4]. A microinjection platform to deliver exogenes into the cell may produce significantly higher expression rates whilst also providing a simplified transformation method which does not necessitate the removal of algal cell walls as required in many current methods [5]. This study describes the initial development of such a device.

The microinjection platform for this study uses a wide-diameter pipette with mild suction to immobilize a cell-wall-intact microalga, then impaling the cell with a second, smaller-diameter pipette filled with genetic material. Implicit with microinjection is a breach of the cell wall and membrane which reduces a cellâ??s viability by exposing the intracellular environment to the extracellular impalement medium. In this study, multiple parameters were controlled and varied to optimize the survival rate of cells during and after impalement. These parameters included the stage of cell growth during which impalement took place, the diameter of the injection pipette, and the medium in which the cells were impaled. Further development of this microinjection platform will include affirming the integrity of the injection pipette after multiple impalements, determining the optimal duration of an impalement, optimizing the voltage used to electroosmotically deliver material into the cell, and developing a medium to be injected into the cells.

References

 

[1] S. P. Mayfield, et al., "Chlamydomonas reinhardtii chloroplasts as protein factories," Current Opinion in Biotechnology, 18, 126-133 (2007).

[2] C. Griesbeck, et al., "Chlamydomonas reinhardtii: A protein expression system for pharmaceutical and biotechnological proteins," Molecular Biotechnology, 34, 213-223 (2006).

[3] J. M. Coll, "Review. Methodologies for transferring DNA into eukaryotic microalgae," Spanish Journal of Agricultural Research, 4, 316-330 (2006).

[4] A. Q. Rao, et al., "The myth of plant transformation," Biotechnology Advances, 27, 753-760 (2009).

[5] A. Crossway and D. Facciotti, "Plant cell microinjection technique". US Patent 4743548, 10 May 1988.