Advances in transgenic rat production.
ABSTRACT 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.
- SourceAvailable from: Charu Ramakrishnan
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- "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). "
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.Neuron 12/2011; 72(5):721-33. DOI:10.1016/j.neuron.2011.10.028 · 15.98 Impact Factor
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- "The methylated or nonmethylated KT2H-CD40Ig transposons were pre-treated with RNAsecure® (Ambion, Austin, TX) and either cut with SspI to linearize or left supercoiled prior to cleanup with the Qiagen MiniPrep Kit (Valencia, CA) and eluted in injection buffer prior to serial dialysis as performed above. The rat transgene was purified using a Nucleospin kit (Clontech, Mountain View, CA) as previously described (Filipiak and Saunders 2006). SB11 mRNA was prepared as using the Ambion (Austin, TX) mMessage mMachine® T3 kit as previously described (Wilber et al. 2006). "
ABSTRACT: Heightened interest in relevant models for human disease increases the need for improved methods for germline transgenesis. We describe a significant improvement in the creation of transgenic laboratory mice and rats by chemical modification of Sleeping Beauty transposons. Germline transgenesis in mice and rats was significantly enhanced by in vitro cytosine-phosphodiester-guanine methylation of transposons prior to injection. Heritability of transgene alleles was also greater from founder mice generated with methylated versus non-methylated transposon. The artificial methylation was reprogrammed in the early embryo, leading to founders that express the transgenes. We also noted differences in transgene insertion number and structure (single-insert versus concatemer) based on the influence of methylation and plasmid conformation (linear versus supercoiled), with supercoiled substrate resulting in efficient transpositional transgenesis (TnT) with near elimination of concatemer insertion. Combined, these substrate modifications resulted in increases in both the frequency of transgenic founders and the number of transgenes per founder, significantly elevating the number of potential transgenic lines. Given its simplicity, versatility and high efficiency, TnT with enhanced Sleeping Beauty components represents a compelling non-viral approach to modifying the mammalian germline.Transgenic Research 03/2010; 20(1):29-45. DOI:10.1007/s11248-010-9386-5 · 2.28 Impact Factor
Article: A brief history of animal modeling.[Show abstract] [Hide abstract]
ABSTRACT: Comparative medicine is founded on the concept that other animal species share physiological, behavioral, or other characteristics with humans. Over 2,400 years ago it was recognized that by studying animals, we could learn much about ourselves. This technique has now developed to the point that animal models are employed in virtually all fields of biomedical research including, but not limited to, basic biology, immunology and infectious disease, oncology, and behavior.Missouri medicine 110(3):201-5.