Novel applications of a modified gene gun: implications for new research in neuroscience
thesis
posted on 2023-07-26, 13:27authored byJohn A. O'Brien
The original Bio-Rad gene gun was unable to transfect acute or organotypic brain slices, as the amount of helium gas used, the distance for the gold-coated microcarriers to travel to target area were not optimised for fragile tissues, such as the brain. Typically, tissues were severely damaged by a helium shock wave and only a few cells were transfected. It was essential to improve gene gun accuracy by restricting the gold particles from being propelled superficially over a wide area. It was also necessary to increase the amount of DNA or dye delivery into intact tissues. Furthermore, for the gene gun to perform successfully on brain slices the helium gas pressure had to be lowered thereby reducing the degree of cell damage incurred during a biolistic delivery. Without knowing it at the time, the modified gene gun had worked particularly well on a variety of other fragile tissues, and not just the brain. However, the modified gun was not optimised for cultured cells as other
transfection methods were available. A particularly notable point of this work was the successful labelling of individual Purkinje dendritic spines from live nerve cells in the cerebellum region of the brain.
Biolistic images of Purkinje cells show that the distribution of dendritic spines are not random
(O’Brien and Unwin, 2006). Spines were shown to grow in elaborate regular linear arrays, that
trace short-pitch helical paths around the dendrites. It was apparent that the spines are arranged
to maximize the probability that the dendritic arbour would interact with any afferent axon. This
was an important discovery as there has been much debate as to how spines develop on a
dendritic shaft. There are three general views to this question, each proposing a theory describing
a model for spinogenesis. Classification of the three models in relation to our findings is
described in chapter six of this thesis.
The Investigation of spine morphology by biolistics was further optimized; gold particles were
reduced from a micrometre to forty nanometres (O’Brien and Lummis, 2011), demonstrating that
it is possible to use gold-coated DNA nanoparticles of this size to transfect tissue revealing
exquisite structural detail. It was possible to observe boutons making synaptic contacts with the
pyramidal nerve spines in the hippocampal region of the brain. The findings so far have shown
spines from the pyramidal shaft are similar to the spines in the cerebellum, forming regular linear
arrays. Recent studies had linked defects in the function of presynaptic boutons to the etiology of
several neurodevelopment and neurodegenerative diseases, including autism and Alzheimer’s
disease. Our discovery could help to understand why there are abnormalities in dendritic spines
which are associated with pathological conditions characterized by cognitive decline, such as
mental retardation, Alzheimer’s, stroke and schizophrenia (Yuste and Bonhoeffer, 2001).
This thesis provides a synthesis of knowledge about biolistic technology. It is presented as a
narrative from improving the gene gun transfection efficiency in brain slices to the development
of nano-biolistics. The delivery of DNA and fluorescent dyes into living cells by biolistic delivery
should enable a detailed map of the anatomical connections between individual cells and groups
of cells to be constructed, providing a “wiring diagram” of connections. The implications of this
are discussed in Chapter twelve.