| Summary: | 碩士 === 國立陽明大學 === 生命科學系暨基因體科學研究所 === 100 === This thesis contains two parts. The first part is to establish the genetic deficiency mouse model of cortactin-binding protein 2 (CTTNBP2). The previous study had shown that loss-of-function CTTNBP2 results in decreased size and density of the dendritic spines in cultured neurons. Since spines are the major locations of excitatory synapses in mammalian brains, CTTNBP2 is likely to play a critical role in brain development and function. To explore the possibility, the conditioned knockout mice of CTTNBP2 were generated by using C57BL/6J embryonic stem cells. The exon 4 of CTTNBP2 genome was flanked with 2 loxP sites, which is able to selectively disrupt the functional CTTNBP2 expression by Cre recombinase. 25 chimeric mice had been obtained, and one of them is capable to transmit C57BL/6J (B6) genome to germline. In the future, the CTTNBP2 knockout mice will be used to investigate the in vivo role of CTTNBP2 in dendritic spinogenesis, and also to explore the physiological functions of CTTNBP2.
The second part is to explore the function of toll-like receptor 7 (TLR7) in neuronal morphogenesis and neurogenesis. TLRs are the critical receptors recognizing both pathogenic and damage-associated molecules and triggering innate immunity. In addition to initiating innate immune response, our previous observations showed that addition of TLR7 agonists reduced axonal and dendritic branches and length in cultured neurons. To investigate the function of TLR7 in vivo, in utero electroporation experiment was performed to knockdown TLR7 at embryonic days 15.5 (E15.5). The morphology of the layer 2/3 neurons of cerebral cortex was then examined after birth. Compared with control, knockdown of TLR7 promoted dendritic arborization of cortical neurons at post-natal days 7 (P7) and P14. Interestingly, the effect of TLR7 knockdown on neuronal morphology was diminished at P21, suggesting that TLR7 is more critical at early postnatal stages. For neurogenesis, there were more dividing neural progenitor cells in TLR7 knockout mice at E18 but not at adult. It also supports a critical role of TLR7 in neurogenesis at early developmental stage. We reasoned that this temporal effect of TLR7 is due to massive cell death during development, which provides the endogenous ligands for TLR7. In conclusion, these in vivo studies provide the evidences supporting the important roles of TLR7 in neuronal morphogenesis and neurogenesis.
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