Browsing by Subject "Mutagenesis, Insertional"
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Item A Novel Transgenic Rat Model for the Study of Germ Cell Biology(2005-08-11) Cronkhite, Jennifer T.; Garbers, David L.With over one million publications in scientific journals, the rat is a very important biological model in science. Unfortunately, since the introduction of genetic manipulation technology in the mouse, extension of this technology to the rat has proven to be very difficult. In an attempt to generate a transgenic line of rats expressing GFP in all cells of the body, a serendipitous integration of a ROSA-EGFP transgene resulted in exclusive expression of EGFP in the germ cells of both sexes. EGFP expression was uniform and robust in cleavage stage embryos beginning at the late 2-cell stage and continuing through blastocyst development where expression became restricted to cells of the inner cell mass. Subsequent analysis showed high EGFP expression exclusively in primordial, embryonic, and adult germ cells. This unique expression pattern makes this EGFP marked locus the first molecular marker of the germline lineage in both sexes in mammals. FISH was used to localize the transgene insertion to chromosome 11q11-q12, proximal to Grik1 and in close proximity to Ncam2. Analysis of the region did not identify known germ cell-specific genes but did identify 19 ESTs or transcribed loci present in testes, ovary, or pre-implantation libraries from mice or rats. The unique germ cell specific expression of EGFP in these transgenic rats makes them an excellent novel tool to study germ cell origin, development, and differentiation. To evaluate the utility of the transgenic line for germ cell transplantation studies, non-selected, freshly isolated seminiferous tubule cells were transferred to the testis of recipient males. The donor cell population colonized the testis at a surprisingly high efficiency within 30 days following transfer. Since EGFP is a vital marker, the colonization process can be followed in vivo and the extent of colonization quantified. This assay was then used to define when developing germ cells first acquire apparent stem cell activity, and to assess the plasticity of adult SP bone marrow cells to enter the germ lineage.Item The Role of SHANK3 at the Synapse and Its Implications in Autism-Associated Behaviors and Synaptic Transmission(2015-04-10) Kouser, Mehreen; Rothenfluh, Adrian; Huber, Kimberly M.; Bibb, James A.; Powell, Craig M.Autism is a neurodevelopmental disorder characterized by an increase in repetitive behaviors and impairments in social interaction and communication. Since its discovery, a multitude of studies have linked SHANK3 to autism. Moreover, deletion of SHANK3 has been shown to cause Phelan McDermid Syndrome (22q13 Deletion Syndrome) by several human studies. Shank3 is a multi domain post-synaptic scaffolding proteins that is found in excitatory synapses and plays a critical role in forming the post-synaptic density by connecting the necessary machinery together. In this study, I have characterized a homozygous Shank3 mutation in mice that deletes exon 21(Shank3ΔC) including the Homer binding domain. In the homozygous state, deletion of exon 21 results in loss of the major, naturally occurring Shank3 protein bands. Shank3ΔC/ΔC mice exhibit an increased localization of mGluR5 to the synapses in the hippocampus, a decrease in NMDA/AMPA excitatory postsynaptic current ratio in area CA1 of hippocampus, reduced long-term potentiation in area CA1, and deficits in hippocampus-dependent spatial learning and memory. In addition, these mice also exhibit motor-coordination deficits, hypersensitivity to heat, novelty avoidance, altered locomotor response to novelty, and minimal social abnormalities. I also report on a novel mouse model of human autism caused by the insertion of a single guanine nucleotide into exon 21 (Shank3G) which causes a premature STOP codon and loss of major higher molecular weight Shank3 isoforms at the synapse like the Shank3ΔC/ΔC mice. Shank3G/G mice exhibit deficits in hippocampus-dependent spatial learning, impaired motor coordination, and altered response to novelty. Shank3G/G mice also exhibit impaired hippocampal excitatory transmission and plasticity. Finally, Shank3G/G mice were designed to be genetically rescued to wild-type at various times during development. In this study, I also report on the biochemical and behavioral results of the genetic rescue in Shank3G/G mice after the completion of neurodevelopment. I was able to achieve a biochemical rescue in the Shank3G/G mice. Interestingly, not all the behavioral impairments observed in Shank3G/G mice were replicated in the Reversible-Shank3G/G mutation mice making the interpretation of the data more challenging which is discussed in detail in this thesis.Item Understanding Autism Pathology: Insights from Genetic Mouse Model Manipulation of KCTD13 and SHANK3(2017-07-19) Ochoa Escamilla, Christine; Konopka, Genevieve; Powell, Craig M.; Huber, Kimberly M.; Eisch, Amelia J.; Tsai, PeterCopy number variations (CNVs) contribute to the etiology of Autism Spectrum Disorders (ASDs). Deletions/duplications in human chromosomal region 16p11.2 are frequently associated with ASD and other neurodevelopmental disorders. Specific genes within this region may be important for determining autism risk, though none has been individually linked to ASD. To understand how single 16p11.2 genes contribute to CNV deletion pathology, we delete a candidate among the 16p11.2 genes, Kctd13, in mice to examine its function in mammalian brain. We report that our Kctd13 deletion mouse model results in decreased synaptic transmission (input/output curves and mEPSC and mIPSC frequency) in area CA1 of the hippocampus with normal paired-pulse ratio, normal mEPSC/IPSC amplitude, and no change in another correlate of presynaptic release probability, suggesting a decrease in the number of functional synapses. Consistent with these results, reduced synaptic transmission correlates with increased levels of RhoA, the KCTD13/CUL3 ubiquitin ligase substrate, and is reversed by RhoA inhibition, confirming increased RhoA as one important molecular mechanism. These changes correlate with a decrease in total dendritic length and spine density in area CA1 of the hippocampus, confirming the decrease in synapse number. This genetic mouse model also displays increased locomotor activity, a robust behavioral phenotype in 16p11.2 deletion mouse models. These results suggest that KCTD13 regulates neuronal morphology and synapse number and likely does so via alteration of RhoA signaling pathways. Together, these data implicate Kctd13 in regulation of neuronal function relevant to neuropsychiatric disorders and reveal a potential role for RhoA as a future preclinical therapeutic target with potential to benefit patients containing KCTD13 deletions. SHANK3 (also known as PROSAP2) functions as a postsynaptic scaffolding protein at excitatory synapses. Mutations and deletions within SHANK3 are known to cause idiopathic autism, Phelan-McDermid (aka 22q13 microdeletion) syndrome, and other neuropsychiatric disorders. We create a novel mouse model of human autism caused by the insertion of a single guanine nucleotide into exon 21 (Shank3G). The resulting frameshift causes a premature STOP codon and loss of major higher molecular weight SHANK3 isoforms at the synapse. At the cellular level, Shank3G/G mice exhibit impaired hippocampal excitatory transmission and plasticity as well as changes in baseline NMDA receptor-mediated synaptic responses. Changes in NMDA receptor function correlate with decreased phosphorylation of the GluN2BR Tyr 1472 site in the Shank3G/G mice. These results identify clear alterations in synaptic function and in the biochemistry of NMDA receptors in a novel, genetically accurate mouse model of autism mimicking an autism-associated insertion mutation. These findings reveal a potential role for NMDA receptors as a preclinical therapeutic target for patients with SHANK3 mutations.