Docosahexaenoic acid normalizes QT interval in long QT type 2 transgenic rabbit models in a genotype-specific fashion.

Aim: Long QT syndrome (LQTS) is a cardiac channelopathy predisposing to ventricular arrhythmias and sudden cardiac death. Since current therapies often fail to prevent arrhythmic events in certain LQTS subtypes, new therapeutic strategies are needed. Docosahexaenoic acid (DHA) is a polyunsaturated fatty acid, which enhances the repolarizing IKs current.

Transgenic LQT2, LQT5, and LQT2-5 rabbit models with decreased repolarisation reserve for prediction of drug-induced ventricular arrhythmias.

Background And Purpose: Reliable prediction of pro-arrhythmic side effects of novel drug candidates is still a major challenge. Although drug-induced pro-arrhythmia occurs primarily in patients with pre-existing repolarisation disturbances, healthy animals are employed for pro-arrhythmia testing. To improve current safety screening, transgenic long QT (LQTS) rabbit models with impaired repolarisation reserve were generated by overexpressing loss-of-function mutations of human HERG (HERG-G628S, loss of I ; LQT2), KCNE1 (KCNE1-G52R, decreased I ; LQT5), or both transgenes (LQT2-5) in the heart.

Glomerulosclerosis in transgenic rabbits with ubiquitous Venus protein expression.

Focal segmental glomerulosclerosis (FSGS) is a potential cause of nephrotic syndrome both in humans and pet mammals. Glomerulopathy was reported earlier in green fluorescent protein (GFP) transgenic (TG) mice, but glomerulosclerosis has not been examined in GFP TG rabbits so far. In the present study, the potential manifestation of FSGS was investigated in both Venus TG rabbits generated by Sleeping Beauty (SB) transposition and age-matched control New Zealand White (NZW) rabbits.

Secretion of a recombinant protein without a signal peptide by the exocrine glands of transgenic rabbits.

Transgenic rabbits carrying mammary gland specific gene constructs are extensively used for excreting recombinant proteins into the milk. Here, we report refined phenotyping of previously generated Venus transposon-carrying transgenic rabbits with particular emphasis on the secretion of the reporter protein by exocrine glands, such as mammary, salivary, tear and seminal glands. The Sleeping Beauty (SB) transposon transgenic construct contains the Venus fluorophore cDNA, but without a signal peptide for the secretory pathway, driven by the ubiquitous CAGGS (CAG) promoter.

Placenta-specific gene manipulation in rabbits.

Lentiviral gene constructs can be efficiently and specifically delivered to trophoblast cell lineages in rodents. In vivo genetic manipulation of trophoblast cell lines enables functional and developmental studies in the placenta. In this report we show that genetic modification can be produced in the extraembryonic tissues of rabbits by lentiviral gene constructs.

Rabbit models as tools for preclinical cardiac electrophysiological safety testing: Importance of repolarization reserve.

It is essential to more reliably assess the pro-arrhythmic liability of compounds in development. Current guidelines for pre-clinical and clinical testing of drug candidates advocate the use of healthy animals/tissues and healthy individuals and focus on the test compound's ability to block the hERG current and prolong cardiac ventricular repolarization. Also, pre-clinical safety tests utilize several species commonly used in cardiac electrophysiological studies.

Transposon-Based Reporter Marking Provides Functional Evidence for Intercellular Bridges in the Male Germline of Rabbits.

The Sleeping Beauty transposon system was established as a robust and efficient method for germline transgenesis in different mammalian species. The generation of transgenic mice, rats, rabbits and swine carrying an identical Venus reporter construct delivered by transposon-mediated gene transfer enables comparative studies of gene expression in these lines of mammalian models. Whereas comparable expression patterns of the Venus reporter were found in somatic tissues, preliminary studies suggested that a striking difference in reporter expression may exist in mature spermatozoa of these species.

Expression of Active Fluorophore Proteins in the Milk of Transgenic Pigs Bypassing the Secretory Pathway.

We describe the expression of recombinant fluorescent proteins in the milk of two lines of transgenic pigs generated by Sleeping Beauty transposon-mediated genetic engineering. The Sleeping Beauty transposon consisted of an ubiquitously active CAGGS promoter driving a fluorophore cDNA, encoding either Venus or mCherry. Importantly, the fluorophore cDNAs did not encode for a signal peptide for the secretory pathway, and in previous studies of the transgenic animals a cytoplasmic localization of the fluorophore proteins was found.

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.

Visualization of Calcium Dynamics in Kidney Proximal Tubules.

Intrarenal changes in cytoplasmic calcium levels have a key role in determining pathologic and pharmacologic responses in major kidney diseases. However, cell-specific delivery of calcium-sensitive probes in vivo remains problematic. We generated a transgenic rat stably expressing the green fluorescent protein-calmodulin-based genetically encoded calcium indicator (GCaMP2) predominantly in the kidney proximal tubules.

Germline transgenesis in rabbits by pronuclear microinjection of Sleeping Beauty transposons.

The laboratory rabbit (Oryctolagus cuniculus) is widely used as a model for a variety of inherited and acquired human diseases. In addition, the rabbit is the smallest livestock animal that is used to transgenically produce pharmaceutical proteins in its milk. Here we describe a protocol for high-efficiency germline transgenesis and sustained transgene expression in rabbits by using the Sleeping Beauty (SB) transposon system.

Germline transgenesis in rodents by pronuclear microinjection of Sleeping Beauty transposons.

We describe a protocol for high-efficiency germline transgenesis and sustained transgene expression in two important biomedical models, the mouse and the rat, by using the Sleeping Beauty transposon system. The procedure is based on co-injection of synthetic mRNA encoding the SB100X hyperactive transposase, together with circular plasmid DNA carrying a transgene construct flanked by binding sites for the transposase, into the pronuclei of fertilized oocytes. Upon translation of the transposase mRNA, enzyme-mediated excision of the transgene cassettes from the injected plasmids followed by permanent genomic insertion produces stable transgenic animals.

NFκB induces overexpression of bovine FcRn: a novel mechanism that further contributes to the enhanced immune response in genetically modified animals carrying extra copies of FcRn.

Among the many functions of the neonatal Fc receptor (FcRn) for IgG, it binds to IgG-opsonized antigen complexes and propagates their traffic into lysosomes where antigen processing occurs. We previously reported that transgenic (Tg) mice and rabbits that carry multiple copies and overexpress FcRn have augmented humoral immune responses. Nuclear factor-kappa B (NFκB) is a critical molecule in the signaling cascade in the immune response.

Low titer lentiviral transgenesis in rodents with simian immundeficiency virus vector.

Efficient production of transgenic animals using low-titer lentiviral constructs remains challenging. Here we demonstrate that microinjection of simian immundeficiency virus-derived lentiviral constructs can produce transgenic mice and rats with high efficiency even when using low-titer virus preparations.

Induced pluripotent stem cells derived from rabbits exhibit some characteristics of naïve pluripotency.

Not much is known about the molecular and functional features of pluripotent stem cells (PSCs) in rabbits. To address this, we derived and characterized 2 types of rabbit PSCs from the same breed of New Zealand White rabbits: 4 lines of embryonic stem cells (rbESCs), and 3 lines of induced PSCs (rbiPSCs) that were obtained by reprogramming adult skin fibroblasts. All cell lines required fibroblast growth factor 2 for their growth and proliferation.

Discovery of pluripotency-associated microRNAs in rabbit preimplantation embryos and embryonic stem-like cells.

MicroRNAs (miRNAs) are small non-coding RNAs that regulate multiple biological processes. Increasing experimental evidence implies an important regulatory role of miRNAs during embryonic development and in embryonic stem (ES) cell biology. In the current study, we have described and analyzed the expression profile of pluripotency-associated miRNAs in rabbit embryos and ES-like cells.

Transposon-mediated transgenesis, transgenic rescue, and tissue-specific gene expression in rodents and rabbits.

Germline transgenesis is an important procedure for functional investigation of biological pathways, as well as for animal biotechnology. We have established a simple, nonviral protocol in three important biomedical model organisms frequently used in physiological studies. The protocol is based on the hyperactive Sleeping Beauty transposon system, SB100X, which reproducibly promoted generation of transgenic founders at frequencies of 50-64, 14-72, and 15% in mice, rats, and rabbits, respectively.

Characterization of the rabbit neonatal Fc receptor (FcRn) and analyzing the immunophenotype of the transgenic rabbits that overexpresses FcRn.

The neonatal Fc receptor (FcRn) regulates IgG and albumin homeostasis, mediates maternal IgG transport, takes an active role in phagocytosis, and delivers antigen for presentation. We have previously shown that overexpression of FcRn in transgenic mice significantly improves the humoral immune response. Because rabbits are an important source of polyclonal and monoclonal antibodies, adaptation of our FcRn overexpression technology in this species would bring significant advantages.

Recombinant protein expression in milk of livestock species.

Producing complex recombinant proteins in the milk of transgenic animals offers several advantages: large amounts of proteins can be obtained, and in most cases, these proteins are properly folded, assembled, cleaved, and glycosylated. The level of expression of foreign genes in the mammalian gland cannot be predicted in all cases, and appropriate vectors must be used. The main elements of these vectors are as follows: a well-characterized specific promoter, the coding region of the gene of interest, preferably with a homologous or heterologous intron, to improve transcription efficiency, and an insulator or boundary element to counteract the chromosomal position effects at the integration site.

Neonatal FcR overexpression boosts humoral immune response in transgenic mice.

The neonatal FcR (FcRn) regulates IgG and albumin homeostasis, mediates maternal IgG transport, takes active part in phagocytosis, and delivers Ag for presentation. We have previously shown that overexpression of FcRn in transgenic (Tg) mice extends the half-life of mouse IgG by reducing its clearance. In this paper, we demonstrate that immunization of these mice with OVA and trinitrophenyl-conjugated human IgG results in a 3- to 10-fold increase of Ag-specific IgM and IgG in serum.

Nociception, neurogenic inflammation and thermoregulation in TRPV1 knockdown transgenic mice.

Transgenic mice with a small hairpin RNA construct interfering with the expression of transient receptor potential vanilloid 1 (TRPV1) were created by lentiviral transgenesis. TRPV1 expression level in transgenic mice was reduced to 8% while the expression of ankyrin repeat domain 1 (TRPA1) was unchanged. Ear oedema induced by topical application of TRPV1 agonist capsaicin was completely absent in TRPV1 knockdown mice.

The miR-290-295 cluster promotes pluripotency maintenance by regulating cell cycle phase distribution in mouse embryonic stem cells.

The mmu-miR-290-295 cluster codes for a family of microRNAs (miRNAs) that are expressed de novo during early embryogenesis and are specific for mouse embryonic stem cells (ESC) and embryonic carcinoma cells (ECC). Detailed sequence analysis and alignment studies of miR-290-295 precursors demonstrated that the cluster has evolved by repeated duplication events of the ancient miR-290 precursor. We show that under serum starvation, overexpression of miR-290-295 miRNAs withhold ES cells from early differentiation, ensures their high proliferation rate and capacity for forming alkaline phosphate positive colonies.

Microsatellite analysis to estimate genetic relationships among five bulgarian sheep breeds.

Herein, genetic relationships among five breeds of Bulgarian sheep were estimated using microsatellite markers. The total number of alleles identified was 226 at the 16 loci examined. D(A) distance values were used for phylogenetic tree construction with the UPGMA algorithm.