دورية
Precise CRISPR-Cas9-mediated mutation of a membrane trafficking domain in the Drosophilavesicular monoamine transporter gene
| العنوان: | Precise CRISPR-Cas9-mediated mutation of a membrane trafficking domain in the Drosophilavesicular monoamine transporter gene |
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| المؤلفون: | Asuncion, James D., Eamani, Aditya, Rohrbach, Ethan W., Knapp, Elizabeth M., Deshpande, Sonali A., Bonanno, Shivan L., Murphy, Jeremy E., Lawal, Hakeem O., Krantz, David E. |
| المصدر: | Current Research in Physiology; January 2023, Vol. 6 Issue: 1 |
| مستخلص: | Monoamine neurotransmitters such as noradrenalin are released from both synaptic vesicles (SVs) and large dense-core vesicles (LDCVs), the latter mediating extrasynaptic signaling. The contribution of synaptic versus extrasynaptic signaling to circuit function and behavior remains poorly understood. To address this question, we have previously used transgenes encoding a mutation in the DrosophilaVesicular Monoamine Transporter (dVMAT) that shifts amine release from SVs to LDCVs. To circumvent the use of transgenes with non-endogenous patterns of expression, we have now used CRISPR-Cas9 to generate a trafficking mutant in the endogenous dVMATgene. To minimize disruption of the dVMATcoding sequence and a nearby RNA splice site, we precisely introduced a point mutation using single-stranded oligonucleotide repair. A predicted decrease in fertility was used as a phenotypic screen to identify founders in lieu of a visible marker. Phenotypic analysis revealed a defect in the ovulation of mature follicles and egg retention in the ovaries. We did not detect defects in the contraction of lateral oviducts following optogenetic stimulation of octopaminergic neurons. Our findings suggest that release of mature eggs from the ovary is disrupted by changing the balance of VMAT trafficking between SVs and LDCVs. Further experiments using this model will help determine the mechanisms that sensitize specific circuits to changes in synaptic versus extrasynaptic signaling. |
| قاعدة البيانات: | Supplemental Index |
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