Transgenic Rabbits Research Articles

Therapeutic immunization with a mixture of herpes simplex virus 1 glycoprotein D-derived “asymptomatic” human CD8+ T-cell epitopes decreases spontaneous ocular shedding in latently infected HLA transgenic rabbits: association with low frequency of local PD-1+ TIM-3+ CD8+ exhausted T cells.

Most blinding ocular herpetic disease is due to reactivation of herpes simplex virus 1 (HSV-1) from latency rather than to primary acute infection. No herpes simplex vaccine is currently available for use in humans. In this study, we used the HLA-A*02:01 transgenic (HLA Tg) rabbit model of ocular herpes to assess the efficacy of a therapeutic vaccine based on HSV-1 gD epitopes that are recognized mainly by CD8(+) T cells from "naturally" protected HLA-A*02:01-positive, HSV-1-seropositive healthy asymptomatic (ASYMP) individuals (who have never had clinical herpes disease). Three ASYMP CD8(+) T-cell epitopes (gD(53-61), gD(70-78), and gD(278-286)) were linked with a promiscuous CD4(+) T-cell epitope (gD(287-317)) to create 3 separate pairs of CD4-CD8 peptides, which were then each covalently coupled to an Nε-palmitoyl-lysine moiety, a Toll-like receptor 2 (TLR-2) ligand. This resulted in the construction of 3 CD4-CD8 lipopeptide vaccines. Latently infected HLA Tg rabbits were immunized with a mixture of these 3 ASYMP lipopeptide vaccines, delivered as eye drops in sterile phosphate-buffered saline (PBS). The ASYMP therapeutic vaccination (i) induced HSV-specific CD8(+) T cells that prevent HSV-1 reactivation ex vivo from latently infected explanted trigeminal ganglia (TG), (ii) significantly reduced HSV-1 shedding detected in tears, (iii) boosted the number and function of HSV-1 gD epitope-specific CD8(+) T cells in draining lymph nodes (DLN), conjunctiva, and TG, and (iv) was associated with fewer exhausted HSV-1 gD-specific PD-1(+) TIM-3+ CD8(+) T cells. The results underscore the potential of an ASYMP CD8(+) T-cell epitope-based therapeutic vaccine strategy against recurrent ocular herpes. Seventy percent to 90% of adults harbor herpes simplex virus 1 (HSV-1), which establishes lifelong latency in sensory neurons of the trigeminal ganglia. This latent state sporadically switches to spontaneous reactivation, resulting in viral shedding in tears. Most blinding herpetic disease in humans is due to reactivation of HSV-1 from latency rather than to primary acute infection. To date, there is no licensed therapeutic vaccine that can effectively stop or reduce HSV-1 reactivation from latently infected sensory ganglia and the subsequent shedding in tears. In the present study, we demonstrated that topical ocular therapeutic vaccination of latently infected HLA transgenic rabbits with a lipopeptide vaccine that contains exclusively human “asymptomatic” CD8(+) T-cell epitopes successfully decreased spontaneous HSV-1 reactivation, as judged by a significant reduction in spontaneous shedding in tears. The findings should guide the clinical development of a safe and effective T-cell-based therapeutic herpes vaccine.

Autocrine Human Urotensin II Enhances Macrophage-Derived Foam Cell Formation in Transgenic Rabbits.

Circulating urotensin II (UII) is involved in the development of atherosclerosis. However, the role of autocrine UII in the development of atherosclerosis remains unclear. Here, we tested the hypothesis that autocrine UII would promote atherosclerosis. Transgenic rabbits were created as a model to study macrophage-specific expressing human UII (hUII) and used to investigate the role of autocrine UII in the development of atherosclerosis. Transgenic rabbits and their nontransgenic littermates were fed a high cholesterol diet to induce atherosclerosis. Comparing the transgenic rabbits with their nontransgenic littermates, it was observed that hUII expression increased the macrophage-positive area in the atherosclerotic lesions by 45% and the positive area ratio by 56% in the transgenic rabbits. Autocrine hUII significantly decreased the smooth muscle cell-positive area ratio in transgenic rabbits (by 54%), without affecting the plasma levels of total cholesterol, triglycerides, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, and glucose and adipose tissue contents. These results elucidated for the first time that autocrine UII plays an important role in the development of atherosclerosis by increasing the accumulation of macrophage-derived foam cell.

Calcium Mediated Mechanism of Early Afterdepolarizations in LQT2 Ventricular Myocytes

Background: Loss of function mutations in hERG potassium channels underlie the long QT syndrome type 2 (LQT2). LQT2 is associated with fatal ventricular arrhythmias promoted by triggered activity in the form of early afterdepolarizations (EADs). However, the cellular mechanism of EAD formation remains unexplored.Methods: We have investigated the mechanism of EAD formation in LQT2 ventricular myocytes using a physiologically detailed computational model of calcium (Ca2+) cycling and membrane voltage dynamics. This model bridges the submicron scale of individual couplons of plasmalemmal L-type Ca2+ channels clusters and sarcoplasmic reticulum (SR) Ca2+ release units (CRUs) and the whole cell. We incorporate the novel experimental finding that ryanodine receptors (RyRs) Ca2+ release channels are remodeled in ventricular mycoytes isolated from LQT2 transgenic rabbits; RyRs are hyperphosphorylated leading to enhanced channel activity and hence increased Ca2+ leak.Results: Computer simulations with RyR hyperactivity modeled as an increased rate of channel opening show that hyperactivity is causally linked to EAD formation. Under stimulation with the β-adrenergic agonist isoproterenol (ISO), LQT2 myocytes with hyperactive RyRs exhibit EADs together with decreased SR load and Ca2+ transient (CaT) amplitude, while myocytes with normal RyR activity exhibit a prolonged action potential without reductions of SR load and CaT amplitude. Simulations show that RyR hyperactivity shortens RyR refractoriness at the CRU level, resulting in late aberrant Ca2+ releases during repolarization. Those releases promote onset of EADs by driving the forward mode Na+-Ca2+ exchanger NCX1 current, which slows repolarization and allows reactivation of L-type Ca2+ current. Modeling predictions are in good agreement with experimental observations, which show that EADs in ISO-stimulated LQT2 myocytes are accompanied by late Ca2+ releases together with decreased SR load and CaT amplitude, and that both late releases and EADs are abolished by inhibition of CaMKII.

Examining the Role of Direct cAMP-Binding Versus PKA-Mediated Effects on Interactions between the Cardiac Potassium Channel α-Subunit Proteins Herg and KvLQT1

KvLQT1 and hERG are the voltage-gated K+ channel α-subunits of the cardiac repolarizing currents IKs and IKr, respectively. These currents function in vivo with some redundancy to maintain appropriate action potential durations (APDs) in cardiomyocytes. As such, protein-protein interactions between hERG and KvLQT1 may be important in normal cardiac electrophysiology, as well as in arrhythmia and sudden cardiac death. Previous phenomenological observations of functional, mutual downregulation between these complementary repolarizing currents in transgenic rabbit models have motivated our investigations into interactions between hERG and KvLQT1. Moreover, reports of interplay between hERG and KvLQT1 have been described both in the literature and anecdotally, and our recent work demonstrated that the interaction is mediated by the C-termini of both α-subunits and is modulated by cAMP. We hypothesize that direct binding of cAMP to the putative cyclic nucleotide binding domain (CNBD) in the hERG C-terminus abrogates hERG-KvLQT1 interactions, as assessed by FRET analyses. We have developed ion channel fusions to GFP variants, which include hERG CNBD mutants as well as phospho-null and phospho-mimetic mutants. cAMP levels are altered in heterologous cells expressing ion channel constructs through membrane-permeable cAMP analogs or forskolin along with IBMX. Through classic biochemical assays and quantitative FRET approaches, we aim to delineate the direct role of cAMP from that of downstream, PKA-mediated effects in regulating interactions between KvLQT1 and hERG in model systems. This work potentially furthers our understanding of hERG-KvLQT1 interactions and may elucidate mechanisms that underlie many types of arrhythmia as well as characterize novel interactions between two distinct potassium channel families.

Transient Outward K+ Current Underlies Heterogeneity of Action Potential Duration and Early Afterdepolarization from Right Ventricle in Transgenic Rabbit Model of Long QT Type 1

Introduction: Long QT syndrome type 1 (LQT1) is a congenital disease lacking slowly activating K+ channel (IKs), associated with polymorphic VT (pVT) and sudden cardiac death. Tissue heterogeneity has been proposed as an important factor to trigger and maintain pVT. We investigated ionic mechanisms that underlie regional differences in the formation of early afterdepolarization (EAD) using transgenic rabbit model of LQT1.Methods: The initiation of pVT under isoproterenol was mapped using optical mapping and myocytes isolated from RV and septum were studied using voltage clamp and confocal calcium imaging.Results: Optical mapping of LQT1 hearts showed pVT preferentially originating from right ventricle (RV) (16 of 18 pVTs), which was changed after perfusing transient outward K channel (Ito) blocker, 0.5 mM 4-aminopyridine (only 1 of 5 pVTs from RV, n=5 hearts). Myocytes isolated from RV demonstrated higher incidence of EADs under 50 nM isoproterenol (8 of 12 cells from RV vs. 2 of 11 cells from septum). Voltage clamp study highlighted regional differences of Ito (4.7 ± 0.5 in RV vs. 2.9 ± 0.7 pA/pF in septum at 0 mV) but other currents such as ICaL, IKr and RyR-mediated Ca2+ leak and SERCA-mediated Ca2+ uptake were same between RV and septum. Computer modeling study of rabbit action potential lacking IKs exhibit frequent EADs but reduction of Ito prevented EAD formation, verifying that Ito plays a major role in EAD formation by providing repolarizing currents during the plateau phase to maintain the membrane potential lower enough to re-activate ICa window current to form EADs.Conclusion: Regional differences of Ito in LQT1 rabbits underlie frequent pVTs originating mostly from RV myocytes with high incidence of EADs.

Modulation of Action Potential Alternans by IKs in Myocardial Infarction

Background: Patients with post myocardial infarction (MI) have increased incidence of ventricular arrhythmias. The slowly activating K+ channels (IKs) has been reported to be downregulated consistently but in different degrees in the post-MI hearts. The role of downregulation of IKs in arrhythmogenesis in post MI remains to be defined.Hypothesis: We hypothesize that significant suppression of IKs in post MI impairs the rate-dependent and Ca2+-dependent augmentation of repolarization reserve resulting in increased alternans and thereby arrhythmias.Methods: We evaluated the effect of IKs on alternans in post MI using transgenic rabbit model of Long QT Type 1 (LQT1) lacking IKs. LQT1 and littermate control (LMC) rabbits underwent MI procedures and optical mapping was performed to record Vm and Ca2+ simultaneously from the 3 week post-MI hearts (n=9 LQT1-MI, 4 LQT-MI Sham, 10 LMC-MI, 9 LMC-Sham hearts).Results: The dispersion of APD was enhanced in LQT1-MI compared to LQT1-Sham and LMC-MI (αAPDmax-min = 28±4 ms vs. 20±3 ms and 27±6 ms). APD alternans was more pronounced in LQT1-MI, accompanied with large amplitude Ca2+ alternans, and often spatially discordant (see traces and restitution curves in panel A&B). Spatially discordant alternans in LQT1-MI was highly dynamic, preceded by sudden appearance of Ca2+ nodal lines near the MI border zone (panel C, dark lines), associated with high VF induction (n=9/9 LQT1-MI vs. 1/4 LQT1-sham and 7/10 LMC-MI).Conclusion: Our results demonstrate the importance of IKs as a repolarization reserve in MI and that significant downregulation of IKs may promote spatially discordant Ca2+ and APD alternans and VF induction.

Methods Introduction of Transgenic Animal

At present, the most common used method for producing transgenic animal is known as DNA microinjection. It directly injects the exogenous gene into fertilized egg cells by using a syringe with micromanipulation instrument. With the exogenous gene integrated into DNA, a transgenic animal is produced. The founders of this method are Jaenisch, Mintz etc. Gordon and Palmiter [1] successively obtained transgenic animals by this method. This method is commonly used at present, and now the transgenic animal research is mostly going based on the Palmiter method, or the method on its improvement. Wang Minhua [2] reported he produced transgenic rabbits with microinjection method transferring anti plague virus nucleic acid enzyme gene. Application of Krimpenfort produced transgenic bovine using both embryos cultured in vitro and microinjection methods.

Abstract 15529: Transgenic Short QT Syndrome Type 1 Rabbits (HERG-N588K) Mimic the Human Phenotype with Shortened Cardiac Repolarization, Ventricular Fibrillation, and Sudden Death

Introduction and Hypothesis: Short QT syndrome is an inherited ion channelopathy with an accelerated cardiac repolarization due to gain-of-function mutations in K+ channel genes. Patients are prone to ventricular tachycardia and sudden cardiac death (SCD). Detailed knowledge about mechanisms of arrhythmogenesis is lacking. We aimed at generating transgenic SQT1 rabbits as novel tool to investigate these mechanisms. Methods: FLAG-HA-tagged HERG-N588K cDNA and the b-MyHC promoter were integrated into a pBSII SK vector and microinjected into rabbit oocytes. SQT1 rabbits and their wildtype (WT) littermates were subjected to in vivo ECG and ex vivo monophasic action potential (AP) measurements. Electrical remodeling was investigated using real-time PCR. Results: We successfully generated 3 transgenic SQT1 founder rabbits. One of the founders died suddenly at the age of 60 days. In SQT1 rabbits (F1) not subjected to any experiments 3/11 died around day 40 vs. 0/8 WT rabbits (p<0.05). SQT1 founder rabbits and their offspring demonstrated a shortened QT interval compared to WT (QT, ms, SQT1, n=44, 147.8±1.7 vs. WT, n=24, 166.4±2.9, p<0.0001, heart-rate corrected QTi, %, 91.6±0.7 vs. 98.1±1.1, p<0.0001) with no gender differences among SQT1 rabbits. Moreover, in SQT1, AP duration was shortened in all segments of LV base, mid, and apex (LV base-lat, APD90, ms, SQT1, n=7, 118.6±5.1 vs. WT, n=9, 154.4±2.2, p<0.0001; APD75, 93.1±6.8 vs. 127.1±1.9, p<0.0001). Ventricular fibrillation (VF) occurred more often during AP measurements in SQT1 than in WT rabbits (SQT1, 5/13 vs. WT, 0/8, p<0.05). FLAG-HA-HERG-N588K channels expressed in HEK cells demonstrated increased IKr steady currents and impaired inactivation indicative of a gain-of-function. Remodeling of ion channels and Ca2+ handling proteins occurred in SQT1 rabbits with a downregulation of L-type Ca2+ channel mRNA in LV septum-mid (SQT1, n=4, 0.84±0.1 vs. WT, n=6, 1.03±0.1, p<0.05) and of RyR2 in LV septum-apex (0.85±0.1 vs. 1.08±0.1, p<0.05). Conclusion: These first transgenic SQT1 rabbits over-expressing a HERG-N588K gain-of-function mutation mimic the human phenotype with shortening of QT and APD, a high rate of VF and SCD. These models are thus a useful tool to explore arrhythmogenic mechanisms in SQTS.

Complex excitation dynamics underlie polymorphic ventricular tachycardia in a transgenic rabbit model of long QT syndrome type 1.

Long QT syndrome type 1 (LQT1) is a congenital disease arising from a loss of function in the slowly activating delayed potassium current IKs, which causes early afterdepolarizations (EADs) and polymorphic ventricular tachycardia (pVT). The purpose of this study was to investigate the mechanisms underlying pVT using a transgenic rabbit model of LQT1. Hearts were perfused retrogradely, and action potentials were recorded using a voltage-sensitive dye and CMOS cameras. Bolus injection of isoproterenol (140 nM) induced pVT initiated by focal excitations from the right ventricle (RV; n = 16 of 18 pVTs). After the pVT was initiated, complex focal excitations occurred in both the RV and the left ventricle, which caused oscillations of the QRS complexes on ECG, consistent with the recent proposal of multiple shifting foci caused by EAD chaos. Moreover, the action potential upstroke in pVT showed a bimodal distribution, demonstrating the coexistence of 2 types of excitation that interacted to produce complex pVT: Na(+) current (INa)-mediated fast conduction and L-type Ca(2+) current (ICa)-mediated slow conduction coexist, manifesting as pVT. Addition of 2 μM tetrodotoxin to reduce INa converted pVT into monomorphic VT. Reducing late INa in computer simulation converted pVT into a single dominant reentry, agreeing with experimental results. Our study demonstrates that pVT in LQT1 rabbits is initiated by focal excitations from the RV and is maintained by multiple shifting foci in both ventricles. Moreover, wave conduction in pVT exhibits bi-excitability, that is, fast wavefronts driven by INa and slow wavefronts driven by ICa co-exist during pVT.

Rabbit models for the study of human atherosclerosis: from pathophysiological mechanisms to translational medicine.

Laboratory animal models play an important role in the study of human diseases. Using appropriate animals is critical not only for basic research but also for the development of therapeutics and diagnostic tools. Rabbits are widely used for the study of human atherosclerosis. Because rabbits have a unique feature of lipoprotein metabolism (like humans but unlike rodents) and are sensitive to a cholesterol diet, rabbit models have not only provided many insights into the pathogenesis and development of human atherosclerosis but also made a great contribution to translational research. In fact, rabbit was the first animal model used for studying human atherosclerosis, more than a century ago. Currently, three types of rabbit model are commonly used for the study of human atherosclerosis and lipid metabolism: (1) cholesterol-fed rabbits, (2) Watanabe heritable hyperlipidemic rabbits, analogous to human familial hypercholesterolemia due to genetic deficiency of LDL receptors, and (3) genetically modified (transgenic and knock-out) rabbits. Despite their importance, compared with the mouse, the most widely used laboratory animal model nowadays, the use of rabbit models is still limited. In this review, we focus on the features of rabbit lipoprotein metabolism and pathology of atherosclerotic lesions that make it the optimal model for human atherosclerotic disease, especially for the translational medicine. For the sake of clarity, the review is not an attempt to be completely inclusive, but instead attempts to summarize substantial information concisely and provide a guideline for experiments using rabbits.

Progesterone modulates SERCA2a expression and function in rabbit cardiomyocytes

We recently showed that progesterone treatment abolished arrhythmias and sudden cardiac death in a transgenic rabbit model of long QT syndrome type 2 (LQT2). Moreover, levels of cardiac sarco(endo)plasmic reticulum Ca(2+)-ATPase type 2a (SERCA2a) were upregulated in LQT2 heart extracts. We hypothesized that progesterone treatment upregulated SERCA2a expression, thereby reducing Ca(2+)-dependent arrhythmias in LQT2 rabbits. We therefore investigated the effect of progesterone on SERCA2a regulation in isolated cardiomyocytes. Cardiomyocytes from neonatal (3- to 5-day-old) rabbits were isolated, cultured, and treated with progesterone and other pharmacological agents. Immunoblotting was performed on total cell lysates and sarcoplasmic reticulum-enriched membrane fractions for protein abundance, and mRNA transcripts were quantified using real-time PCR. The effect of progesterone on baseline Ca(2+) transients and Ca(2+) clearance was determined using digital imaging. Progesterone treatment increased the total pool of SERCA2a protein by slowing its degradation. Using various pharmacological inhibitors of degradation pathways, we showed that progesterone-associated degradation of SERCA2a involves ubiquitination, and progesterone significantly decreases the levels of ubiquitin-tagged SERCA2a polypeptides. Our digital imaging data revealed that progesterone significantly shortened the decay and duration of Ca(2+) transients. Progesterone treatment increases protein levels and activity of SERCA2a. Progesterone stabilizes SERCA2a, in part, by decreasing the ubiquitination level of SERCA2a polypeptides.

Hyperphosphorylation of RyRs underlies triggered activity in transgenic rabbit model of LQT2 syndrome.

Loss-of-function mutations in human ether go-go (HERG) potassium channels underlie long QT syndrome type 2 (LQT2) and are associated with fatal ventricular tachyarrhythmia. Previously, most studies focused on plasma membrane-related pathways involved in arrhythmogenesis in long QT syndrome, whereas proarrhythmic changes in intracellular Ca(2+) handling remained unexplored. We investigated the remodeling of Ca(2+) homeostasis in ventricular cardiomyocytes derived from transgenic rabbit model of LQT2 to determine whether these changes contribute to triggered activity in the form of early after depolarizations (EADs). Confocal Ca(2+) imaging revealed decrease in amplitude of Ca(2+) transients and sarcoplasmic reticulum Ca(2+) content in LQT2 myocytes. Experiments using sarcoplasmic reticulum-entrapped Ca(2+) indicator demonstrated enhanced ryanodine receptor (RyR)-mediated sarcoplasmic reticulum Ca(2+) leak in LQT2 cells. Western blot analyses showed increased phosphorylation of RyR in LQT2 myocytes versus controls. Coimmunoprecipitation experiments demonstrated loss of protein phosphatases type 1 and type 2 from the RyR complex. Stimulation of LQT2 cells with β-adrenergic agonist isoproterenol resulted in prolongation of the plateau of action potentials accompanied by aberrant Ca(2+) releases and EADs, which were abolished by inhibition of Ca(2+)/calmodulin-dependent protein kinase type 2. Computer simulations showed that late aberrant Ca(2+) releases caused by RyR hyperactivity promote EADs and underlie the enhanced triggered activity through increased forward mode of Na(+)/Ca(2+) exchanger type 1. Hyperactive, hyperphosphorylated RyRs because of reduced local phosphatase activity enhance triggered activity in LQT2 syndrome. EADs are promoted by aberrant RyR-mediated Ca(2+) releases that are present despite a reduction of sarcoplasmic reticulum content. Those releases increase forward mode Na(+)/Ca(2+) exchanger type 1, thereby slowing repolarization and enabling L-type Ca(2+) current reactivation.

Pronounced effects of HERG-blockers E-4031 and erythromycin on APD, spatial APD dispersion and triangulation in transgenic long-QT type 1 rabbits.

BackgroundProlongation of action potential duration (APD), increased spatial APD dispersion, and triangulation are major factors promoting drug-induced ventricular arrhythmia. Preclinical identification of HERG/IKr-blocking drugs and their pro-arrhythmic potential, however, remains a challenge. We hypothesize that transgenic long-QT type 1 (LQT1) rabbits lacking repolarizing IKs current may help to sensitively detect HERG/IKr-blocking properties of drugs.MethodsHearts of adult female transgenic LQT1 and wild type littermate control (LMC) rabbits were Langendorff-perfused with increasing concentrations of HERG/IKr-blockers E-4031 (0.001–0.1 µM, n = 9/7) or erythromycin (1–300 µM, n = 9/7) and APD, APD dispersion, and triangulation were analyzed.ResultsAt baseline, APD was longer in LQT1 than in LMC rabbits in LV apex and RV mid. Erythromycin and E-4031 prolonged APD in LQT1 and LMC rabbits in all positions. However, erythromycin-induced percentaged APD prolongation related to baseline (%APD) was more pronounced in LQT1 at LV base-lateral and RV mid positions (100 µM, LQT1, +40.6±9.7% vs. LMC, +24.1±10.0%, p<0.05) and E-4031-induced %APD prolongation was more pronounced in LQT1 at LV base-lateral (0.01 µM, LQT1, +29.6±10.6% vs. LMC, +19.1±3.8%, p<0.05) and LV base-septal positions. Moreover, erythromycin significantly increased spatial APD dispersion only in LQT1 and increased triangulation only in LQT1 in LV base-septal and RV mid positions. Similarly, E-4031 increased triangulation only in LQT1 in LV apex and base-septal positions.ConclusionsE-4031 and erythromycin prolonged APD and increased triangulation more pronouncedly in LQT1 than in LMC rabbits. Moreover, erythromycin increased APD dispersion only in LQT1, indicating that transgenic LQT1 rabbits could serve as sensitive model to detect HERG/IKr-blocking properties of drugs.

Induction of body weight loss through RNAi-knockdown of APOBEC1 gene expression in transgenic rabbits.

In the search of new strategies to fight against obesity, we targeted a gene pathway involved in energy uptake. We have thus investigated the APOB mRNA editing protein (APOBEC1) gene pathway that is involved in fat absorption in the intestine. The APOB gene encodes two proteins, APOB100 and APOB48, via the editing of a single nucleotide in the APOB mRNA by the APOBEC1 enzyme. The APOB48 protein is mandatory for the synthesis of chylomicrons by intestinal cells to transport dietary lipids and cholesterol. We produced transgenic rabbits expressing permanently and ubiquitously a small hairpin RNA targeting the rabbit APOBEC1 mRNA. These rabbits exhibited a moderately but significantly reduced level of APOBEC1 gene expression in the intestine, a reduced level of editing of the APOB mRNA, a reduced level of synthesis of chylomicrons after a food challenge, a reduced total mass of body lipids and finally presented a sustained lean phenotype without any obvious physiological disorder. Interestingly, no compensatory mechanism opposed to the phenotype. These lean transgenic rabbits were crossed with transgenic rabbits expressing in the intestine the human APOBEC1 gene. Double transgenic animals did not present any lean phenotype, thus proving that the intestinal expression of the human APOBEC1 transgene was able to counterbalance the reduction of the rabbit APOBEC1 gene expression. Thus, a moderate reduction of the APOBEC1 dependent editing induces a lean phenotype at least in the rabbit species. This suggests that the APOBEC1 gene might be a novel target for obesity treatment.

High incidence of spontaneous cataracts in aging laboratory rabbits of an inbred strain.

To investigate the occurrence of spontaneous cataracts in a breeding colony of the inbred EIII/JC strain of New Zealand White rabbits (Oryctolagus cuniculi) and the congenic strain of EIII/JC-HLA-A2.1transgenic rabbits. A retrospective study was conducted by collecting and analyzing data from clinical records for individual rabbits filed between January 2011 and October 2013. Thirteen cases (eight females and five males) of cataract were identified in a group of 51 EIII/JC inbred rabbits with a morbidity of 25.5%. The median age of the rabbits identified with unilateral or bilateral cataracts was 43 months in contrast to the median age of 23 months of the entire group of 51 rabbits. Additionally, seven cases (five females and two males) of cataracts were identified in a group of 21 EIII/JC-HLA-A2.1 transgenic rabbits. The EIII/JC-HLA-A2.1 transgenic rabbits showed similar morbidity (33.3%) and median age (41 months) for the development of cataracts as the EIII/JC rabbits. In both groups, none of the rabbits younger than 37 months developed cataracts while 13 (93%) of 14 EIII/JC rabbits aged 37-49 months and seven (63.6%) of 11 EIII/JC-HLA-A2.1 transgenic rabbits aged 37-43 months developed cataracts. In contrast, none of 78 outbred rabbits with a median age of 26 months (10-67 months) developed cataracts. Results of this study indicate that the occurrence and high incidence of spontaneous cataracts in this inbred strain (EIII/JC) of rabbits were strictly age related and consistently transmitted through inbreeding.

Efficient generation of sFat-1 transgenic rabbits rich in n-3 polyunsaturated fatty acids by intracytoplasmic sperm injection.

N-3 polyunsaturated fatty acids (n-3 PUFAs) have their first double bond at the third carbon from the methyl end of the fatty-acid chain and had been proven to be beneficial to human health. However, mammals cannot produce n-3 PUFAs by themselves because they lack the n-3 fatty-acid desaturase (Fat-1) gene. Thus, the possibility of producing sFat-1 transgenic rabbits was explored in this study. The transgenic cassette of pPGK1-sFat-1-CMV-EGFP was constructed and transgenic rabbit embryos were produced by intracytoplasmic sperm injection (ICSI). When 123 EGFP-positive embryos at the 2-8-cell stage were transplanted into the oviduct of four oestrous-synchronised recipients, two of them became pregnant and gave birth to seven pups. However, transfer of embryos into the uterus of oestrous-synchronised recipients and oviduct or uterus of oocyte donor rabbits did not result in pregnancy. The integration of the sFat-1 gene was confirmed in six of the seven live pups by PCR and Southern blot. The expression of the sFat-1 gene in the six transgenic pups was also detected by reverse transcription polymerase chain reaction (RT-PCR). Gas chromatography-mass spectrometry analysis revealed that transgenic rabbits exhibited an ~15-fold decrease in the ratio of n-6:n-3 PUFAs in muscle compared with wild-type rabbits and non-transgenic rabbits. These results demonstrate that sFat-1 transgenic rabbits can be produced by ICSI and display a low ratio of n-6:n-3 PUFAs.

The effect of mammary gland-specific transgene expression on rabbit reproductive gland structure.

Transgenic rabbits are excellent animal models for human diseases and suitable bioreactors for the production of recombinant proteins on an experimental and commercial scale. The aim of this study was to compare the structure of the mWAP-hFVIII transgenic and non-transgenic rabbit ovarian and testicular tissue. Ovarian and testicular tissue samples were taken from transgenic and non-transgenic New Zealand White rabbits, examined by optical microscopy and analyzed morphometrically. An increase of the relative volume of primary follicles and a decrease of the relative volume of antral follicles was detected in the transgenic ovarian structure (P < 0.05), but other developmental follicular stages and follicular diameters were not affected (P > 0.05). In the testes a significant decrease (P < 0.05) of the epithelial height was detected in the transgenic testicular structure, but the relative volume of all basic structures (germinal epithelium, interstitium and lumen) was unaltered (P > 0.05). Generally, this study demonstrates a weak negative effect of mWAP-hFVIII transgenesis on rabbit gonadal structure.

Inhibition of Glycosphingolipid Synthesis Ameliorates Atherosclerosis and Arterial Stiffness in Apolipoprotein E −/− Mice and Rabbits Fed a High-Fat and -Cholesterol Diet

Glycosphingolipids, integral components of the cell membrane, have been shown to serve as messengers, transducing growth factor-initiated phenotypes. Here, we have examined whether inhibition of glycosphingolipid synthesis could ameliorate atherosclerosis and arterial stiffness in transgenic mice and rabbits. Apolipoprotein E(-/-) mice (12 weeks of age; n=6) were fed regular chow or a Western diet (1.25% cholesterol, 2% fat). Mice were fed 5 or 10 mg/kg of an inhibitor of glycosphingolipid synthesis, D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP), solubilized in vehicle (5% Tween-80 in PBS); the placebo group received vehicle only. At 20 and 36 weeks of age, serial echocardiography was performed to measure aortic intima-media thickening. Aortic pulse-wave velocity measured vascular stiffness. Feeding mice a Western diet markedly increased aortic pulse-wave velocity, intima-media thickening, oxidized low-density lipoprotein, Ca(2+) deposits, and glucosylceramide and lactosylceramide synthase activity. These were dose-dependently decreased by feeding D-PDMP. In liver, D-PDMP decreased cholesterol and triglyceride levels by raising the expression of SREBP2, low-density lipoprotein receptor, HMGCo-A reductase, and the cholesterol efflux genes (eg, ABCG5, ABCG8). D-PDMP affected very-low-density lipoprotein catabolism by increasing the gene expression for lipoprotein lipase and very-low-density lipoprotein receptor. Rabbits fed a Western diet for 90 days had extensive atherosclerosis accompanied by a 17.5-fold increase in total cholesterol levels and a 3-fold increase in lactosylceramide levels. This was completely prevented by feeding D-PDMP. Inhibition of glycosphingolipid synthesis ameliorates atherosclerosis and arterial stiffness in apolipoprotein E(-/-) mice and rabbits. Thus, inhibition of glycosphingolipid synthesis may be a novel approach to ameliorate atherosclerosis and arterial stiffness.

Lp(a)/apo(a) Modulate MMP-9 Activation and Neutrophil Cytokines in Vivo in Inflammation to Regulate Leukocyte Recruitment

Lipoprotein(a) [Lp(a)] is an independent risk factor for cardiovascular diseases, but the mechanism is unclear. The pathogenic risk of Lp(a) is associated with elevated plasma concentration, small isoforms of apolipoprotein [apo(a)], the unique apolipoprotein of Lp(a), and a mimic of plasminogen. Inflammation is associated with both the initiation and recovery of cardiovascular diseases, and plasminogen plays an important role in leukocyte recruitment. Because Lp(a)/apo(a) is expressed only in primates, transgenic mice were generated, apo(a)tg and Lp(a)tg mice, to determine whether Lp(a)/apo(a) modifies plasminogen-dependent leukocyte recruitment or whether apo(a) has an independent role in vivo. Plasminogen activation was markedly reduced in apo(a)tg and Lp(a)tg mice in both peritonitis and vascular injury inflammatory models, and was sufficient to reduce matrix metalloproteinase-9 activation and macrophage recruitment. Furthermore, neutrophil recruitment and the neutrophil cytokines, CXCL1/CXCL2, were suppressed in apo(a)tg mice in the abdominal aortic aneurysm model. Reconstitution of CXCL1 or CXCL2 restored neutrophil recruitment in apo(a)tg mice. Apo(a) in the plasminogen-deficient background and Lp(a)tg mice were resistant to inhibition of macrophage recruitment that was associated with an increased accumulation of apo(a) in the intimal layer of the vessel wall. These data indicate that, in inflammation, Lp(a)/apo(a) suppresses neutrophil recruitment by plasminogen-independent cytokine inhibition, and Lp(a)/apo(a) inhibits plasminogen activation and regulates matrix metalloproteinase-9 activation and macrophage recruitment.

Effective gene targeting in rabbits using RNA-guided Cas9 nucleases

Effective gene targeting in rabbits using RNA-guided Cas9 nucleases