Ear Skin Biopsies Research Articles

340PRODUCTION OF TRANSGENIC PORCINE BLASTOCYSTS BY HANDMADE CLONING

The present study demonstrates the application of the recently developed handmade cloning (HMC) technique in production of transgenic porcine blastocysts. The HMC technique was originally established for bovine nuclear transfer (Vajta et al., 2003, Biol. Reprod. 68, 571–578), and has the advantages of being less demanding and more productive than traditional nuclear transfer techniques. Cumulus-oocyte complexes were aspirated from slaughterhouse ovaries and matured for 41h. Subsequently, the cumulus cells were removed by pipetting in 1mgmL−1 hyaluronidase in HEPES-buffered TCM-199; zonae pellucidae were removed by incubation in 2mgmL−1 pronase in HEPES-buffered TCM-199 supplemented with 2% cattle serum (T2) for 1min. Bisection was performed by hand under a stereomicroscope using a microblade in 5μgmL−1 cytochalasin B in TCM-199 supplemented with 20% cattle serum (T20). Demi-oocytes were incubated in 5μgmL−1 Hoechst 33342 in T20 for 10min, followed by examination under UV light to select the halves containing no chromatin, i.e., the cytoplasts. Porcine fibroblasts harvested from an ear skin biopsy were transfected with pN1-EGFP (Clontech) using Lipofectamine (Gibco, Life Technologies). G418 selection (0.8mgmL−1) was applied 48h after transfection, and well separated G418-resistant cell colonies originating from a single transfected cell were isolated, expanded, and cryopreserved. Days before, nuclear transfer cells were grown to a confluent monolayer in DMEM supplemented with 10% FCS. Fusions were performed 43h after start of maturation. One cytoplast was attached to one fibroblast in 500μgmL−1 phytohemagglutinin dissolved in T2. In the fusion chamber, covered with fusion medium (0.3M mannitol, 0.1mM MgSO4, 0.05mM CaCl2, and 0.01% PVA), one cytoplast-fibroblast pair was fused with one cytoplast in a single step. The fusions were performed with a double DC pulse of 65V, each pulse for 20μs and 0.1s apart from each other. Successfully fused embryos were activated 1h after the end of fusion by incubation in 2μM calcium ionophore A23187 in T20 for 5min followed by 3-h incubation in microdrops of culture medium (NCSU-23 with 4mgmL BSA) containing 2mM 6-dimethylaminopurine. Activated embryos were cultured individually in microdrops of culture medium for 7 days. In four independent experiments, 93% of attempted reconstructed embryos fused and survived activation (31/31, 15/23, 28/28, and 37/37, respectively). On Day 7 after activation, the blastocyst rates (per successfully reconstructed embryos) were 6% (2/31), and 7% (1/15), 7% (2/28), and 3% (1/37), respectively. Green Fluorescent Protein was expressed in all cells of the developing blastocysts. The results show that transgenic porcine blastocysts can be produced using HMC, and the technique may also be applied for the production of transgenic pigs.

33DEVELOPMENT OF BOVINE EMBRYOS CLONED WITH FETAL FIBROBLASTS ARRESTED AT G0/G1 PHASE BY DIFFERENT TREATMENTS

Numerous factors have an effect on the development of cloned embryos, and one of the most important might be the synchronization between donor nuclei and recipient ooplasts. The objective of this study was to examine the effect of donor cell treatments for G0/G1 synchronization and the donor cell type on development and incidence of apoptosis in cloned cattle embryos. Primary cultures were established from a female fetus on Day 50 of gestation and adult ear skin biopsies. The cells were used for assessements of cell cycle and apoptosis, and for nuclear transfer. Cells were randomly allocated into 3 experimental treatment groups after 6–8 passages: Group 1 (confluent), cells were cultured in DMEM supplemented with 10% FBS until 90% confluent; Group 2 (serum-starvation), cells were cultured in DMEM supplemented with 0.5% FBS for 5 days; Group 3 (Roscovitine), cells were cultured in DMEM supplemented with 10% FBS and 30μM Roscovitine for 12h. Cell cycle and apoptosis were analyzed using flow cytometry after labelling with DAPI and YO-PRO-1, respectively. At 19h post-maturation (hpm), enucleated oocytes were reconstructed with donor cells and fused by a single DC pulse (1.6kV/cm, 60μs) delivered by a BTX 200. After activation with the combination of ionomycin (5μM, 5min) and cycloheximide (10μgmL−1, 5h), the eggs were cultured in CR1aa medium for 3 days and additionally cultured in CR1aa medium supplemented with 30mgmL−1 BSA for 5 days at 39°C in a humidified atmosphere of 5% CO2 in air. Differences between groups were analyzed using one-way ANOVA after arc-sine transformation of the proportional data. There were no significantly differences in the incidence of cells arrested at G0/G1 for fetal fibroblasts cultured in the three treatment groups (87%, 83% and 80%; confluent, serum starvation and Roscovitine, respectively). More cells were apoptotic in Group 2 compared to the cells in Groups 1 and 3 (12% v. 6 and 6%, respectively) (P<0.05). Blastocyst development of cloned embryos was significantly (P<0.05) higher when fetal fibroblasts from Group 1 were used, compared to Groups 2 and 3 (35.1%, v. 31 and 29.7%, respectively). Similar results were observed in the use of ear skin fibroblasts as nuclear transfer donor cells (32.7%, v. 24 and 24%, respectively). These results suggest that fetal fibroblasts can be effectively synchronized at G0/G1 by three different treatments, including growth to confluence, serum-starvation and Roscovitine treatment. However, based on blastocyst development and levels of apoptosis, the use of confluent fetal fibroblasts as donor cells is more effective than using cells synchronized by serum-starvation or Roscovitine treatment in the production of cloned bovine embryos. [Supported by High Technology Development Project for Agriculture and Forestry Korea, MAF-SGRP, 30012-05-3-SB010 and Cho-A Pharm. Co. LTD.]

Early detection of Borrelia burgdorferi sensu lato infection in Balb/c mice by co-feeding Ixodes ricinus ticks

AbstractIn Europe, Borrelia burgdorferi is transmitted by Ixodes ricinus to animals and human. When infected and uninfected ticks co-feed on a host, spirochetes are transmitted from ticks to animal and also to uninfected ticks. Here, we used uninfected ticks to co-feed with infected ticks on mice to evaluate this method to detect early infection in mice. A total of 128 mice were challenged by infected nymphs placed in capsules glued on the back of the mice. Three days later uninfected larvae were added in the capsule to co-feed with infected nymphs and were examined for Borrelia infection after natural detachment. Infection in mice was also determined by xenodiagnosis and by spirochete isolation from ear skin biopsy and back skin biopsy taken at the tick attachment site one month after infection. A total of 111 mice were found to be infected by at least one of these four methods. Borrelia infection was observed in 95% of mice by the co-feeding method, in 92% of mice by xenodiagnosis, in 69% and in 68% of mice by cultivation of ear and back skin biopsies, respectively. Our results demonstrate that the co-feeding method is a very sensitive method which can be used to detect very early infection in mice infected by tick bites.

Transmission of Borrelia afzelii from Apodemus mice and Clethrionomys voles to Ixodes ricinus ticks: differential transmission pattern and overwintering maintenance.

This study deals with the ecology of Lyme borreliosis in Europe. The relationships between Borrelia burgdorferi sensu lato spirochetes, Clethrionomys and Apodemus rodent reservoirs and the Ixodes ricinus tick vector were investigated during 16 consecutive months in an enzootic area in Switzerland. Cultivation of ear skin biopsies was used to isolate spirochetes from C. glareolus, A. sylvaticus, A. flavicollis and Glis glis. Borrelia infection was more frequently observed in Clethrionomys than in Apodemus. Tick xenodiagnosis was used to determine the infectivity of rodents. The infection rate in ticks fed on Clethrionomys was higher than that in ticks fed on Apodemus, but Apodemus yielded more infected ticks than Clethrionomys because of a better tick moulting success. Xenodiagnostic ticks were placed into BSK medium to obtain isolates. Isolates from rodents and rodent-feeding ticks were all identified as B. afzelii. The follow-up of the infectivity status of repeatedly recaptured rodents clearly demonstrated that these hosts remained infective for ticks during winter till the following spring. Comparing C. glareolus and A. sylvaticus, each rodent species showed different host infection, different host infectivity and contributed differently to the moulting success of feeding ticks. These factors influence differentially the pattern of transmission of B. afzelii from Clethrionomys voles and Apodemus mice to I. ricinus ticks.

EXPERIMENTAL INFECTION OF THE RACCOON (PROCYON LOTOR) WITH BORRELIA BURGDORFERI

The reservoir competence of the raccoon (Procyon lotor) for the Lyme disease spirochete (Borrelia burgdorferi) was evaluated in the laboratory during September 1991 to April 1993. Five raccoons were exposed to spirochete-infected (JD1 and Wisconsin 210 Wise strains) Ixodes scapularis nymphs (20/raccoon). A second feeding of spirochete-infected (Wisconsin 210 Wise strain) nymphs (20/raccoon) was performed with four of the original raccoons. Xenodiagnosis with cohorts of I. scapularis larvae (300/cohort) or nymphs (150/cohort) that were periodically placed on each animal was used to detect infection. We examined 1943 engorged ticks by a indirect immunofluorescence monoclonal antibody assay, but no spirochetes were detected. After exposure to spirochete-infected ticks, blood samples were collected at approximately weekly intervals and ear-skin biopsy samples were taken from each animal every third week. These tissues were placed in Barbour-Stoenner-Kelly media. Spirochetes were isolated in cultures of skin (wk 3, 5, 9, 81, and 83) and blood (wk 5, 8, 9, 11, and 12) of one raccoon and the skin (wk 28 and 31) of another raccoon. Antibody response of each animal was monitored through enzyme-linked immunosorbent assays and immunoblotting of blood serum against B. burgdorferi proteins. Except for one animal, raccoons did not have an antibody response until they were fed upon by a second cohort of infected I. scapularis nymphs. Based on Western blot analyses, raccoons exposed to B. burgdorferi via tick bite responded to the 31- (OspA) and 34-KDa (OspB) antigens. Response to other antigens varied among raccoons. Based on our results raccoons may be inefficient reservoirs for B. burgdorferi. Although some raccoons can become infected with B. burgdorferi, they may not transfer the infection to attached ticks.

SIMULTANEOUS OCCURRENCE OF REJECTION AND GRAFT-VERSUS-HOST REACTION AFTER ALLOGENEIC SMALL BOWEL TRANSPLANTATION

Small bowel transplantation may be complicated not only by rejection but also by graft-versus-host reaction (GVHR). So far, little is known about the association between these two immunological reactions, partly because of the lack of standardized, reproducible experimental models for such studies. In this work, a rat model in which GVHR and rejection occur simultaneously was established. When transplanting small bowel grafts from BN donors to Lewis recipients and thereafter treating the grafts locally with the immunomodulating substance LS-2616, a clinically visible GVHR occurred on day 6, at the same time the first signs of rejection became visible. The GVHR was confirmed by immunohistochemical staining for MHC class II-positive cells in liver and ear skin biopsy specimens. An obvious quantitative difference in the number of positive cells in both organs was observed when local treatment was compared with oral LS-2616 treatment or with findings in organs from untreated animals. We conclude that GVHR and rejection are not mutually exclusive and thus may occur simultaneously, and that this pharmacological model might facilitate further studies of the impact of GVHR on graft rejection and recipient survival.

Immobilization-induced stress decreases lipogenesis in sebaceous glands as well as plasma testosterone levels in male Syrian hamsters

The effects of immobilization-induced stress on plasma testosterone levels and lipogenesis in the sebaceous gland were examined in male Syrian hamsters. To induce immobilization stress, the animals were placed in the prone position and wrapped with flexible steel wire gauze at room temperature. Plasma testosterone levels were determined by radioimmunoassay (RIA) of blood samples and sebaceous lipogenesis was determined by measurement of the incorporation of a lipid precursor, 14C-acetate, in ear skin biopsy samples. Both specimens were obtained immediately after the hamsters were decapitated. Immobilization stress for 4 consecutive days produced a marked fall in plasma testosterone levels and sebaceous lipogenesis. These reductions were reversible, the decreased plasma testosterone levels and decreased sebaceous lipogenesis recovering to the nonstressed or prestimulus levels after approximately 1 week. In 4-day stressed animals in occurrence with a decrease in plasma testosterone, sebaceous lipogenesis in ear with topical application of a small dose of testosterone, which have no effect on plasma testosterone level, was equivalent to that in nonstressed animals. These findings indicated that immobilization-induced stress lowered testosterone secretion, and consequently the testosterone levels in the skin, resulting in decreased lipogenesis in the skin. These results thus suggest that psychological or physiological stress can influence cutaneous function by inducing changes in the neuroendocrine system.