Applications of and considerations for using CRISPR-Cas9-mediated gene conversion systems in rodents.

Genetic elements that are inherited at super-Mendelian frequencies could be used in a 'gene drive' to spread an allele to high prevalence in a population with the goal of eliminating invasive species or disease vectors. We recently demonstrated that the gene conversion mechanism underlying a CRISPR-Cas9-mediated gene drive is feasible in mice. Although substantial technical hurdles remain, overcoming these could lead to strategies that might decrease the spread of rodent-borne Lyme disease or eliminate invasive populations of mice and rats that devastate island ecology. Perhaps more immediately achievable at moderate gene conversion efficiency, applications in a laboratory setting could produce complex genotypes that reduce the time and cost in both dollars and animal lives compared with Mendelian inheritance strategies. Here, we discuss what we have learned from early efforts to achieve CRISPR-Cas9-mediated gene conversion, potential for broader applications in the laboratory, current limitations, and plans for optimizing this potentially powerful technology.

Comparisons of the antibody repertoires of a humanized rodent and humans by high throughput sequencing.

The humanization of animal model immune systems by genetic engineering has shown great promise for antibody discovery, tolerance studies and for the evaluation of vaccines. Assessment of the baseline antibody repertoires of unimmunized model animals will be useful as a benchmark for future immunization experiments. We characterized the heavy chain and kappa light chain antibody repertoires of a model animal, the OmniRat, by high throughput antibody sequencing and made use of two novel datasets for comparison to human repertoires. Intra-animal and inter-animal repertoire comparisons reveal a high level of conservation in antibody diversity between the lymph node and spleen and between members of the species. Multiple differences were found in both the heavy and kappa chain repertoires between OmniRats and humans including gene segment usage, CDR3 length distributions, class switch recombination, somatic hypermutation levels and in features of V(D)J recombination. The Inference and Generation of Repertoires (IGoR) software tool was used to model recombination in VH regions which allowed for the quantification of some of these differences. Diversity estimates of the OmniRat heavy chain repertoires almost reached that of humans, around two orders of magnitude less. Despite variation between the species repertoires, a high frequency of OmniRat clonotypes were also found in the human repertoire. These data give insights into the development and selection of humanized animal antibodies and provide actionable information for use in vaccine studies.

Next Generation Transgenic Rat Model Production.

The next generation of new genetically engineered rat models by microinjection is described. Genome editors such as CRISPR/Cas9 have greatly increased the efficiency with which the rat genome can be modified to generate research models for biomedical research. Pronuclear microinjection of transgene DNA into rat zygotes results in random multicopy transgene integration events that use exogenous promoters to drive expression. Best practices in transgenic animal design indicate the use of precise single copy transgene integration in the genome. This ideal can be achieved by repair of CRISPR/Cas9 chromosome breaks by homology directed repair. The most effective way to achieve this type of transgenic rat model is to deliver genome modification reagents to rat zygotes by pronuclear microinjection. The keys to success in this process are to obtain fertilized eggs (zygotes) from the rat strain of choice, to purify the microinjection reagents, to deliver the reagents to the eggs by pronuclear microinjection, to use the surgical transfer of microinjected eggs to pseudopregnant rats to obtain G0 founder animals that carry the novel genetic modification. Ultimately the success of new rat models is measured by changes in gene expression as in the expression of a new reporter protein such as eGFP, Cre recombinase, or other protein of interest.

Endoscopic Transplantation of Mesenchymal Stem Cell Sheets in Experimental Colitis in Rats.

Owing to the recent progress in regenerative medicine technology, clinical trials that harnessed the regeneration and immune modulation potentiality of stem cells for treating IBD have shown promising results. We investigated the feasibility and utility of intraluminal endoscopic transplantation of rat MSC sheets in murine models of experimental colitis for targeted delivery of stem cells to lesions. We isolated adipose-derived mesenchymal stem cells (AD-MSC) and bone marrow-derived mesenchymal stem cells (BM-MSC) from EGFP-transgenic rats and fabricated the cells in sheet forms using temperature-responsive culture dishes. The MSC sheets were endoscopically transplanted to the inflamed area in electrocoagulation and DNBS colitis model. The effect of the transplantation was verified using endoscopic scoring and histological analysis. In the electrocoagulation model, the AD-MSC group showed significantly decreased ulcer size in the transplanted regions. In the DNBS colitis model, the AD-MSC group showed decreased inflammation and colitis in the transplanted regions. Histologic analysis showed that the MSC sheets had successfully attached to the inflamed mucosa in both the electrocoagulation and DNBS colitis model. Our results show that endoscopic transplantation of MSC sheets could be a new effective mode of stem cell therapy for IBD treatment.

Long Evans rat spermatogonial lines are effective germline vectors for transgenic rat production.

Long Evans rat strains are applied as research models in a broad spectrum of biomedical fields (>15,800 citations, NCBI PubMed). Here, we report an approach to genetically modify the Long Evans rat germline in donor spermatogonial stem cells. Long Evans rat spermatogonial lines were derived from freshly isolated laminin-binding spermatogonia. Laminin-binding spermatogonia were cultured over multiple passages on fibroblast feeder layers in serum-free culture medium containing GDNF and FGF2. Long Evans rat spermatogonial lines were genetically modified by transposon transduction to express a germline, tdTomato reporter gene. Donor rat spermatogonial lines robustly regenerated spermatogenesis after transplantation into testes of busulfan-treated, allogenic, Long Evans rats. Donor-derived spermatogenesis largely restored testis size in the chemically sterilized, recipient Long Evans rats. Recipient Long Evans rats stably transmitted the tdTomato germline marker to subsequent generations. Overall, Long Evans rat spermatogonial lines provided effective donor germline vectors for genetically modifying Long Evans rats.

New transgenic ALS/FTD models on the rat-walk: An Editorial Highlight for 'Increased Ubqln2 expression causes neuron death in transgenic rats'.

This Editorial highlights a study by Huang and colleagues in the current issue of Journal of Neurochemistry. The authors introduce a novel ALS-FTD (amyotrophic lateral sclerosis-frontotemporal dementia) rat model to explore the role of the UBLQN2 gene that has previously been associated with familial ALS-FTD. Over-expression of ubiquilin 2 in the cortex (CTX) and hippocampus of the rat results in ubiquilin 2 aggregates and neurodegeneration together with cognitive deficits. The new rat model not only gives insight into potential molecular underpinnings of ALS-FTD, but also represents an important new tool for future research and therapeutic approaches. Read the highlighted article 'Increased Ubqln2 expression causes neuron death in transgenic rats' on page 285.

A Novel Model of Intravital Platelet Imaging Using CD41-ZsGreen1 Transgenic Rats.

Platelets play pivotal roles in both hemostasis and thrombosis. Although models of intravital platelet imaging are available for thrombosis studies in mice, few are available for rat studies. The present effort aimed to generate fluorescent platelets in rats and assess their dynamics in a rat model of arterial injury. We generated CD41-ZsGreen1 transgenic rats, in which green fluorescence protein ZsGreen1 was expressed specifically in megakaryocytes and thus platelets. The transgenic rats exhibited normal hematological and biochemical values with the exception of body weight and erythroid parameters, which were slightly lower than those of wild-type rats. Platelet aggregation, induced by 20 µM ADP and 10 µg/ml collagen, and blood clotting times were not significantly different between transgenic and wild-type rats. Saphenous arteries of transgenic rats were injured with 10% FeCl3, and the formation of fluorescent thrombi was evaluated using confocal microscopy. FeCl3 caused time-dependent increases in the mean fluorescence intensity of injured arteries of vehicle-treated rats. Prasugrel (3 mg/kg, p.o.), administered 2 h before FeCl3, significantly inhibited fluorescence compared with vehicle-treated rats (4.5 ± 0.4 vs. 14.9 ± 2.4 arbitrary fluorescence units at 30 min, respectively, n = 8, P = 0.0037). These data indicate that CD41-ZsGreen1 transgenic rats represent a useful model for intravital imaging of platelet-mediated thrombus formation and the evaluation of antithrombotic agents.

Transgenic rat model of childhood-onset dermatitis by overexpressing telomerase reverse transcriptase (TERT).

Childhood-onset dermatitis is one of the most common skin disorders in children. Although various mouse models that mirror aspects of dermatitis have become available, there is still a need for an animal model that develops dermatitis in childhood and is more suitable for performing tissue transplantation experiments. There is emerging evidence that peripheral blood T lymphocytes from patients with dermatitis have significantly increased telomerase activity. Here, we developed telomerase reverse transcriptase (TERT)-expressing transgenic (Tg) rats that spontaneously developed eczematous skin inflammation in childhood. Newborn TERT-Tg rats developed visible dermatitis in 56 % of cases, and the skin lesions microscopically showed spongiosis and acanthosis with infiltration of lymphocytes, eosinophils and mast cells. TERT-Tg rats with dermatitis exhibited increased CD4 (2.5-fold) and CD8 (fivefold) T cell numbers compared with dermatitis-free TERT-Tg rats. Stronger TERT activity was observed in the peripheral lymphocytes of dermatitis-positive TERT-Tg rats than those of dermatitis-free TERT-Tg rats. RT-PCR analysis revealed that IL-4 was markedly elevated in the spleen of dermatitis-positive TERT-Tg rats, and that interferon-gamma was increased in the dermatitis lesions. Moreover, skin grafting of TERT-Tg rats with dermatitis onto T cell-deficient nude rats demonstrated that the inflamed skin lesions could not be maintained. Taken together, the results suggest that TERT activation in T lymphocytes is one of the potential predisposing factors for dermatitis. Moreover, our results demonstrated that the TERT-Tg rats mirror aspects of human childhood-onset dermatitis and that these animals represent a potential animal model system for studying childhood-onset dermatitis.

Generation of a Homozygous Transgenic Rat Strain Stably Expressing a Calcium Sensor Protein for Direct Examination of Calcium Signaling.

In drug discovery, prediction of selectivity and toxicity require the evaluation of cellular calcium homeostasis. The rat is a preferred laboratory animal for pharmacology and toxicology studies, while currently no calcium indicator protein expressing rat model is available. We established a transgenic rat strain stably expressing the GCaMP2 fluorescent calcium sensor by a transposon-based methodology. Zygotes were co-injected with mRNA of transposase and a CAG-GCaMP2 expressing construct, and animals with one transgene copy were pre-selected by measuring fluorescence in blood cells. A homozygous rat strain was generated with high sensor protein expression in the heart, kidney, liver, and blood cells. No pathological alterations were found in these animals, and fluorescence measurements in cardiac tissue slices and primary cultures demonstrated the applicability of this system for studying calcium signaling. We show here that the GCaMP2 expressing rat cardiomyocytes allow the prediction of cardiotoxic drug side-effects, and provide evidence for the role of Na(+)/Ca(2+) exchanger and its beneficial pharmacological modulation in cardiac reperfusion. Our data indicate that drug-induced alterations and pathological processes can be followed by using this rat model, suggesting that transgenic rats expressing a calcium-sensitive protein provide a valuable system for pharmacological and toxicological studies.

Recent progress in the genetics of spontaneously hypertensive rats.

The spontaneously hypertensive rat (SHR) is the most widely used animal model of essential hypertension and accompanying metabolic disturbances. Recent advances in sequencing of genomes of BN-Lx and SHR progenitors of the BXH/HXB recombinant inbred (RI) strains as well as accumulation of multiple data sets of intermediary phenotypes in the RI strains, including mRNA and microRNA abundance, quantitative metabolomics, proteomics, methylomics or histone modifications, will make it possible to systematically search for genetic variants involved in regulation of gene expression and in the etiology of complex pathophysiological traits. New advances in manipulation of the rat genome, including efficient transgenesis and gene targeting, will enable in vivo functional analyses of selected candidate genes to identify QTL at the molecular level or to provide insight into mechanisms whereby targeted genes affect pathophysiological traits in the SHR.

Production of fat-1 transgenic rats using a post-natal female germline stem cell line.

Germline stem cell lines possess the abilities of self-renewal and differentiation, and have been established from both mouse and human ovaries. Here, we established a new female germline stem cell (FGSC) line from post-natal rats by immunomagnetic sorting for Fragilis, which showed a normal karyotype, high telomerase activity, and a consistent gene expression pattern of primordial germ cells after 1 year of culture. Using an in vitro differentiation system, the FGSC line could differentiate into oocytes. After liposome-based transfection with green fluorescent protein (GFP) or fat-1 vectors, the FGSCs were transplanted into the ovaries of infertile rats. The transplanted FGSCs underwent oogenesis, and the rats produced offspring carrying the GFP or fat-1 transgene after mating with wild-type male rats. The efficiency of gene transfer was 27.86-28.00%, and 2 months was needed to produce transgenic rats. These findings have implications in biomedical research and potential applications in biotechnology.

A novel reporter rat strain that expresses LacZ upon Cre-mediated recombination.

The recent widespread application of Cre/loxP technology has resulted in a new generation of conditional animal models that can better recapitulate many salient features of human disease. These models benefit from the ability to monitor the expression and functionality of Cre protein. We have generated a conditional (Cre/loxP dependent) LacZ reporter rat (termed the LacZ541 rat) to monitor Cre in transgenic rats. When LacZ541 rats were bred with another transgenic rat line expressing Cre recombinase under the control of the CAG promoter, LacZ/Cre double transgenic embryos displayed ubiquitous expression of LacZ, and when LacZ541 rats were bred with transgenic rats expressing Cre/loxP-dependent oncogenic H- or K-ras, LacZ was expressed in the lesions resulting from the activation of the oncogene. The LacZ541 rat enables evaluation of the performance of Cre-expressing systems which are based upon transgenic rats or somatic gene transfer vectors and provides efficient and simple lineage marking.

Insertional mutagenesis by a hybrid piggyBac and sleeping beauty transposon in the rat.

A hybrid piggyBac/Sleeping Beauty transposon-based insertional mutagenesis system that can be mobilized by simple breeding was established in the rat. These transposons were engineered to include gene trap sequences and a tyrosinase (Tyr) pigmentation reporter to rescue the albinism of the genetic background used in the mutagenesis strategy. Single-copy transposon insertions were transposed into the rat genome by co-injection of plasmids carrying the transposon and RNA encoding piggyBac transposase into zygotes. The levels of transgenic Tyr expression were influenced by chromosomal context, leading to transgenic rats with different pigmentation that enabled visual genotyping. Transgenic rats designed to ubiquitously express either piggyBac or Sleeping Beauty transposase were generated by standard zygote injection also on an albino background. Bigenic rats carrying single-copy transposons at known loci and transposase transgenes exhibited coat color mosaicism, indicating somatic transposition. PiggyBac or Sleeping Beauty transposase bigenic rats bred with wild-type albino rats yielded offspring with pigmentation distinct from the initial transposon insertions as a consequence of germline transposition to new loci. The germline transposition frequency for Sleeping Beauty and piggyBac was ∼10% or about one new insertion per litter. Approximately 50% of the insertions occurred in introns. Chimeric transcripts containing endogenous and gene trap sequences were identified in Gabrb1 mutant rats. This mutagenesis system based on simple crosses and visual genotyping can be used to generate a collection of single-gene mutations in the rat.

Imaging of endocrine gene expression in a humanized transgenic rat.

Reporter gene imaging has revealed cyclical patterns of gene expression in living cells. Transgenic animal studies show that these patterns are modified by tissue architecture.

A new model for facial nerve research: the novel transgenic Thy1-GFP rat.

OBJECTIVE: To introduce a Thy1-GFP transgenic rat model, whose axons constitutively express green fluorescent protein (GFP), in order to study facial nerve regeneration. Facial nerve injury can cause devastating physical and social sequelae. The functional recovery of the facial nerve can result in synkinesis and permanent axonal misrouting. Facial nerve research has been hindered by the lack of available animal models and reliable outcome measures. METHODS: Transgenic Thy1-GFP rats underwent a proximal facial nerve crush injury and were imaged at 0, 1, 2, 4, and 8 weeks after injury. Nerve regeneration was assessed via confocal imaging and fluorescence microscopy. RESULTS: Uninjured animals reliably demonstrated facial nerve fluorescence and had predictable anatomical landmarks. Fluorescence microscopy demonstrated the loss and reappearance of fluorescence with regeneration of axons following injury. This was confirmed with the visualization of denervation and reinnervation of zygomaticus muscle motor end plates using confocal microscopy. CONCLUSIONS: The Thy1-GFP rat is a novel transgenic tool that enables direct visualization of facial nerve regeneration after injury. The utility of this model extends to a variety of clinical facial nerve injury paradigms.

alpha-Synuclein- and MPTP-generated rodent models of Parkinson's disease and the study of extracellular striatal dopamine dynamics: a microdialysis approach.

The classical animal models of Parkinson's disease (PD) rely on the use of neurotoxins, including 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), 6-hydroxydopamine and, more recently, the agricultural chemicals paraquat and rotenone, to deplete dopamine (DA). These neurotoxins elicit motor deficits in different animal species although MPTP fails to induce a significant dopaminergic neurodegeneration in rats. In the attempt to better reproduce the key features of PD, in particular the progressive nature of neurodegeneration, alternative PD models have been developed, based on the genetic and neuropathological links between -synuclein ( -syn) and PD. In vivo microdialysis was used to investigate extracellular striatal DA dynamics in MPTP- and -syn-generated rodent models of PD. Acute and sub-acute MPTP intoxication of mice both induce prolonged release of striatal DA. Such DA release may be considered the first step in MPTP-induced striatal DA depletion and nigral neuron death, mainly through reactive oxygen species generation. Although MPTP induces DA reduction, neurochemical and motor recovery starts immediately after the end of treatment, suggesting that compensatory mechanisms are activated. Thus, the MPTP mouse model of PD may be unsuitable for closely reproducing the features of the human disease and predicting potential long-term therapeutic effects, in terms of both striatal extracellular DA and behavioral outcome. In contrast, the -syn-generated rat model of PD does not suffer from a massive release of striatal DA during induction of the nigral lesion, but rather is characterized by a prolonged reduction in baseline DA and nicotine-induced increases in dialysate DA levels. These results are suggestive of a stable nigrostriatal lesion with a lack of dopaminergic neurochemical recovery. The -syn rat model thus reproduces the initial stage and slow development of PD, with a time-dependent impairment in motor function. This article will describe the above experimental PD models and demonstrate the utility of microdialysis for their characterization.

New lines of GFP transgenic rats relevant for regenerative medicine and gene therapy.

Adoptive cell transfer studies in regenerative research and identification of genetically modified cells after gene therapy in vivo require unequivocally identifying and tracking the donor cells in the host tissues, ideally over several days or for up to several months. The use of reporter genes allows identifying the transferred cells but unfortunately most are immunogenic to wild-type hosts and thus trigger rejection in few days. The availability of transgenic animals from the same strain that would express either high levels of the transgene to identify the cells or low levels but that would be tolerant to the transgene would allow performing long-term analysis of labelled cells. Herein, using lentiviral vectors we develop two new lines of GFP-expressing transgenic rats displaying different levels and patterns of GFP-expression. The "high-expresser" line (GFP(high)) displayed high expression in most tissues, including adult neurons and neural precursors, mesenchymal stem cells and in all leukocytes subtypes analysed, including myeloid and plasmacytoid dendritic cells, cells that have not or only poorly characterized in previous GFP-transgenic rats. These GFP(high)-transgenic rats could be useful for transplantation and immunological studies using GFP-positive cells/tissue. The "low-expresser" line expressed very low levels of GFP only in the liver and in less than 5% of lymphoid cells. We demonstrate these animals did not develop detectable humoral and cellular immune responses against both transferred GFP-positive splenocytes and lentivirus-mediated GFP gene transfer. Thus, these GFP-transgenic rats represent useful tools for regenerative medicine and gene therapy.

Design of expression cassettes for the generation of transgenic animals (including insulators).

The use of transgenesis is relatively rare in rats, and this is because of the relative difficulty in adding foreign genes by the conventional methods. Gene knock out and knock in by the conventional techniques of homologous recombination remain difficult in rats. This situation would be less crucial if the gene constructs were more reliable for the expression of foreign genes. The present chapter describes the state of the art in vector design for various genetic modifications in rats.