Predictable and reproducible production of transgenic rats from a standardized input of egg donors and egg recipients is essential for routine rat model production. In the course of establishing a transgenic rat service, transgenic founders were produced from three transgenes in outbred Sprague-Dawley (SD) rats and four transgenes in inbred Fischer 344 (F344) rats. Key parameters that affect transgenesis efficiency were assessed, including superovulation treatments, methods to prepare pseudopregnant recipients, and microinjection technique. Five superovulation regimens were compared and treatment with 20 IU PMSG and 30 IU HCG was selected for routine use. Four methods to prepare pseudopregnant egg recipients were compared and estrus synchronization with LHRHa and mating to vasectomized males was selected as most effective. More than 80% of eggs survived microinjection when modified pronuclear microinjection needles and DNA buffers were used. The efficiencies of transgenic production in rats and C57BL/6J (B6J) mice were compared to provide a context for assessing the difficulty of transgenic rat production. Compared to B6J mice, SD rat transgenesis required fewer egg donors per founder, fewer pseudopregnant egg recipients per founder, and produced more founders per eggs microinjected. Similar numbers of injection days were required to produce founders. These results suggest that SD rat transgenesis can be more efficient than B6J mouse transgenesis with the appropriate technical refinements. Advances in transgenic rat production have the potential to increase access to rat models.
"But the main experimental advantage of the mouse over the rat is the larger number of available transgenic lines that enable cell-type specificity in neurophysiological experiments (Huang and Zeng, 2013). Although, there are now techniques for manipulating the genome of both species (Filipiak and Saunders, 2006; Witten et al., 2011), the mouse has had a long head start as illustrated by the thousands of transgenic lines available from The Jackson Laboratory repository. "
[Show abstract][Hide abstract] ABSTRACT: Two opposing constraints exist when choosing a model organism for studying the neural basis of adaptive decision-making: (1) experimental access and (2) behavioral complexity. Available molecular and genetic approaches for studying neural circuits in the mouse fulfill the first requirement. In contrast, it is still under debate if mice can perform cognitive tasks of sufficient complexity. Here we compare learning and performance of mice and rats, the preferred behavioral rodent model, during an acoustic flexible categorization two-alternative choice task. The task required animals to switch between two categorization definitions several times within a behavioral session. We found that both species achieved similarly high performance levels. On average, rats learned the task faster than mice, although some mice were as fast as the average rat. No major differences in subjective categorization boundaries or the speed of adaptation between the two species were found. Our results demonstrate that mice are an appropriate model for the study of the neural mechanisms underlying adaptive decision-making, and suggest they might be suitable for other cognitive tasks as well.
Frontiers in Systems Neuroscience 09/2014; 8:173. DOI:10.3389/fnsys.2014.00173
"Injected zygotes were incubated over night in an incubator at 37°C, 5% CO2 and 95% humidity. Morphologically intact one-cell and two-cell embryos were transferred the next day into the oviducts of day 0.5 pseudopregnant (LEW x WKY) F1 rats . "
[Show abstract][Hide abstract] ABSTRACT: Background
Engineered zinc-finger nucleases (ZFN) represented an innovative method for the genome manipulation in vertebrates. ZFN introduced targeted DNA double strand breaks (DSB) and initiated non-homologous end joining (NHEJ) after pronuclear or cytoplasmatic microinjection into zygotes. Resulting frame shift mutations led to functional gene ablations in zebra fish, mice, pigs and also in laboratory rats. Therefore, we targeted the rat Rag1 gene essential for the V(D)J recombination within the immunoglobulin production process and for the differentiation of mature B and T lymphocytes to generate an immunodeficient rat model in the LEW/Ztm strain.
After microinjection of Rag1 specific ZFN mRNAs in 623 zygotes of inbred LEW/Ztm rats 59 offspring were born from which one carried a 4 bp deletion. This frame shift mutation led to a premature stop codon and a subsequently truncated Rag1 protein confirmed by the loss of the full-length protein in Western Blot analysis. Truncation of the Rag1 protein was characterized by the complete depletion of mature B cells. The remaining T cell population contained mature CD4+/CD3+/TCRαβ+ as well as CD8+/CD3+/TCRαβ+ positive lymphocytes accompanied by a compensatory increase of natural killer cells in the peripheral blood. Reduction of T cell development in Rag1 mutant rats was associated with a hypoplastic thymus that lacked follicular structures. Histological evaluation also revealed the near-complete absence of lymphocytes in spleen and lymph nodes in the immunodeficient Rag1 mutant rat.
The Rag1 mutant rat will serve as an important model for transplantation studies. Furthermore, it may be used as a model for reconstitution experiments related to the immune system, particularly with respect to different populations of human lymphocytes, natural killer cells and autoimmune phenomena.
"When combined with optogenetics, these tools now enable selective control of neuromodulatory function with exceptional temporal precision in genetically-defined subpopulations and their projections, and we expect this approach to be readily generalizable to other cell types in rats. This approach capitalizes on BAC technology that had been developed for the generation of transgenic mice (Gong et al., 2007); coupling these constructs with recent advances in pronuclear injection technology in rats (Filipiak and Saunders, 2006) results in a versatile approach that will enable targeting of a virtually unlimited array of geneticallydefined cell-types of interest. Our success in achieving cell-type specific expression in rats was fundamentally related to the very large regulatory/promoter element that we employed (the BACs allowed for a regulatory region of 200–300 kB), which contrasts with the much smaller promoter regions that typically can be packaged in viruses (typically 2–5 kB promoter region, depending on the type of virus and the size of the proteins being expressed by the virus). "
[Show abstract][Hide abstract] ABSTRACT: Currently there is no general approach for achieving specific optogenetic control of genetically defined cell types in rats, which provide a powerful experimental system for numerous established neurophysiological and behavioral paradigms. To overcome this challenge we have generated genetically restricted recombinase-driver rat lines suitable for driving gene expression in specific cell types, expressing Cre recombinase under the control of large genomic regulatory regions (200-300 kb). Multiple tyrosine hydroxylase (Th)::Cre and choline acetyltransferase (Chat)::Cre lines were produced that exhibited specific opsin expression in targeted cell types. We additionally developed methods for utilizing optogenetic tools in freely moving rats and leveraged these technologies to clarify the causal relationship between dopamine (DA) neuron firing and positive reinforcement, observing that optical stimulation of DA neurons in the ventral tegmental area (VTA) of Th::Cre rats is sufficient to support vigorous intracranial self-stimulation (ICSS). These studies complement existing targeting approaches by extending the generalizability of optogenetics to traditionally non-genetically-tractable but vital animal models.
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