Inactivating ZF-Foxq1b with molecular scissors
Patrick Breen, 2013
Zebrafish (Danio rerio) were used to explore the role of the gene Foxq1b, a gene that
is evolutionarily conserved and seems to play an important role during embryonic
development. A Zinc Finger Nuclease (ZFN) was designed using Context-dependant
Assembly (CoDA) to produce a double stranded break in the open read frame of Foxq1b.
Then Non-Homologous End Joining (NHEJ), a natural low fidelity repair mechanism,
ensured that small insertions or deletions were introduced to the cut site during the repair
process. These deletions or insertions in turn produce a stochastic frameshift
mutation leading to an early stop codon, effectively silencing gene function in effected cells.
Our initial results seem to indicate that the ZFN was successful in producing a mosaic knock
out (KO) phenotype in line with what we would expect. We are currently in the process of
reproducing our results, verifying the KO using a molecular or sequencing basis, and further
investigating the role of Foxq1b in the zebrafish model.
Figure 1. Wild type (WT) zebrafish are compared against Foxq1b mosaics at 120 hours post
fertilization (hpf). Fish in the left column have their skeletal systems died with Alcian Blue to
help illustrate the abnormal patterning of the skeletal system. In both columns though, many
abnormal patterning events appear to have occurred. One of the most striking is the fact that
mosaics are cyclopic. This is indicative of abnormal sonic hedgehog signaling which implies
that foxq1b is upstream of the sonic hedgehog signaling pathway.
Faculty Mentor: Antonio Planchart PhD, Mount Desert Island Biological Laboratory
Funded by: INBRE