Timothy Locke '14 - Testing !C31 Integrase Towards Generating a Transgenic GFP Expressing Gryllus bimaculatus
The cricket (Gryllus bimaculatus) is a peculiar model system because it exhibits a high level of neuronal plasticity in adulthood. In a healthy cricket, the auditory interneuron, ascending neuron 2 (AN-2), grows up to, but not over, the midline of the prothoracic ganglion. However, in response to deafferenting one side of the ganglion, AN-2 exhibits compensatory dendritic growth over the midline and forms synapses with the intact afferent neuron from the contralateral side (Horch, 2009). Currently, the technique to visualize AN-2, backfilling the individual nerve with biocytin dye, is inefficient. As an eventual goal, we would like to generate a transgenic cricket that expresses green fluorescent protein (GFP) in such a way that the AN-2 neuron morphology can be easily visualized through confocal microscopy. Previous research by Dr. Noji and colleagues showed transgenic G. bimaculatus GFP expression to be possible through the piggybac transposase enzyme. Unfortunately, genes inserted with piggybac can be excised out of the genome if the piggybac enzyme is reintroduced, therefore we would not be able to do further transgenic modifications using piggybac after initial transgenesis. As a solution, we plan to incorporate integrase-mediated cassette exchange (IMCE) sites using !C31 integrase. !C31 integrase has been shown to function in Drosophila melanogaster (Bateman, 2006) and Bombyx mori (Yonemura, 2012), but it has not yet been tested in crickets. We plan to test this by synthesizing !C31 integrase mRNA and injecting it with a plasmid containing !C31 integrase recognition sites (AttP & AttB). If the integrase functions properly, the plasmid will be cut in two and we can confirm that !C31 integrase is a viable transgenesis tool to use towards making a transgenic cricket.
This summer was spent understanding the !C31 integrase system, practicing injecting cricket eggs, synthesizing mRNA, and cloning the aforementioned vectors. We used two plasmids: one containing the coding gene for !C31 integrase (PHS62) and one containing the !C31 integrase attP and attB sites (PRT504). DHC! competent E. coli cells were transformed with these plasmids and grown up on an agar plate with antibacterial (ampicillin for PHS62 and kanamycin for PRT504) and then a colony was picked from the plate and grown up in LB broth containing the same antibacterial. The resulting cells were stored as bacterial stocks. The plasmids were then extracted and used as templates to make sequencing reactions to confirm the identity of our bacterial stocks. I will perform the injections to test the !C31 integrase system in crickets using the injection protocol used in the Noji lab this fall when I continue this project as an honors project.
In addition to the project with the !C31 integrase, I have also been furthering the research of the recently graduated Noah Pyles on making a ubiquitously expressing GFP transgenic cricket. Noah previously ligated the GFP coding gene downstream of an actin protein promoter and also did the same downstream of the promoter for heat shock protein 70 (HSP70). Both actin and HSP70 have been shown to be ubiquitously expressed in all cricket tissue, so if the transgenesis is successful, the GFP should also be expressed in all cricket tissue. This summer I started to get the full genetic sequence of Noah’s plasmids by setting up sequencing reactions.
Faculty Mentor: Hadley Horch
Funded by the Paller Neuroscience Research Fellowship