Bypassing Glutamic Acid Decarboxylase 1 (Gad1) Induced
Craniofacial Defects with a Photoactivatable Translation Blocker Morpholino
Version 2 2018-10-19, 23:52
Version 1 2018-09-24, 15:40
Posted on 2018-10-19 - 23:52
γ-Amino
butyric acid (GABA) mediated signaling is critical
in the central and enteric nervous systems, pancreas, lungs, and other
tissues. It is associated with many neurological disorders and craniofacial
development. Glutamic acid decarboxylase (GAD) synthesizes GABA from
glutamate, and knockdown of the gad1 gene results
in craniofacial defects that are lethal in zebrafish. To bypass this
and enable observation of the neurological defects resulting from
knocking down gad1 expression, a photoactivatable
morpholino oligonucleotide (MO) against gad1 was
prepared by cyclization with a photocleavable linker rendering the
MO inactive. The cyclized MO was stable in the dark and toward degradative
enzymes and was completely linearized upon brief exposure to 405 nm
light. In the course of investigating the function of the ccMOs in
zebrafish, we discovered that zebrafish possess paralogous gad1 genes, gad1a and gad1b. A gad1b MO injected at the 1–4 cell stage
caused severe morphological defects in head development, which could
be bypassed, enabling the fish to develop normally, if the fish were
injected with a photoactivatable, cyclized gad1b MO
and grown in the dark. At 1 day post fertilization (dpf), light activation
of the gad1b MO followed by observation at 3 and
7 dpf led to increased and abnormal electrophysiological brain activity
compared to wild type animals. The photocleavable linker can be used
to cyclize and inactivate any MO, and represents a general strategy
to parse the function of developmentally important genes in a spatiotemporal
manner.
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O’Connor, Matthew
J.; Beebe, Lindsey L.; Deodato, Davide; Ball, Rebecca E.; Page, A. Tyler; VanLeuven, Ariel J.; et al. (2018). Bypassing Glutamic Acid Decarboxylase 1 (Gad1) Induced
Craniofacial Defects with a Photoactivatable Translation Blocker Morpholino. ACS Publications. Collection. https://doi.org/10.1021/acschemneuro.8b00231
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AUTHORS (11)
MO
Matthew
J. O’Connor
LB
Lindsey L. Beebe
DD
Davide Deodato
RB
Rebecca E. Ball
AP
A. Tyler Page
AV
Ariel J. VanLeuven
KH
Kyle T. Harris
SP
Sungdae Park
VH
Vani Hariharan
JL
James D. Lauderdale
TD
Timothy M. Dore
KEYWORDS
GABA405 nm light1 day post fertilizationgad 1 expressionGlutamic acid decarboxylaseparalogous gad 1 geneselectrophysiological brain activityphotocleavable linkerPhotoactivatable Translation Blocker Morpholino γ- Amino butyric acidcyclized gad 1b MOgad 1 gene resultsGADgad 1b MOphotoactivatable morpholino oligonucleotide