A dual fluorescent multiprobe assay for prion protein genotyping in sheep

BACKGROUND: Scrapie and BSE belong to a group of fatal, transmissible, neurodegenerative diseases called TSE. In order to minimize the risk of natural scrapie and presumed natural BSE in sheep, breeding programmes towards TSE resistance are conducted in many countries based on resistance rendering PRNP polymorphisms at codons 136 (A/V), 154 (R/H) and 171 (R/H/Q). Therefore, a reliable, fast and cost-effective method for routine PRNP genotyping in sheep, applicable in standard equipped molecular genetic laboratories, will be a vital instrument to fulfill the need of genotyping hundreds or thousands of sheep. METHODS: A dual fluorescent multiprobe assay consisting of 2 closed tube PCR reactions containing respectively 4 and 3 dual-labelled fluorescent ASO probes for the detection in real-time of the 7 allelic variants of sheep PRNP mentioned above. RESULTS: The assay is succesfully performed using unpurified DNA as a template for PCR, without any post-PCR manipulations and with semi-automatic determination of the PRNP genotypes. The performance of the assay was confirmed via PCR-RFLP and sequencing in a cross-validation study with 50 sheep. CONCLUSIONS: We report the development and validation of a robust, reliable and reproducible method for PRNP genotyping of a few to many sheep samples in a fast, simple and cost-effective way, applicable in standard equipped molecular genetic laboratories. The described primer/probe design strategy can also be applied for the detection of other polymorphisms or disease causing mutations

Effects of hypodontia on craniofacial structures and mandibular growth pattern

Introduction This study was performed to examine craniofacial structures in persons with hypodontia and to reveal any differences, that may occur, when agenetic teeth are only found in the maxilla, the mandible or in both jaws. The groups consistent of 50 children (33 girls, 17 boys) aged between 9 and 13.5 years were analyzed and assigned to three subgroups. Group 1= upper jaw hypodontia. Group 2= lower jaw hypodontia. Group 3= hypodontia in both jaws. Material and methods Eleven angular and three index measurements from lateral encephalographs and two linear measurements from dental blaster casts were calculated. All data was statistically analyzed, parameters with p<5% were investigated for each subgroup respectively. Results In comparison with standards the study group showed bimaxillary retrognathism and a reduction of the lower anterior facial height. Moreover both overbite and overjet significantly increased. Other values laid within the normal ranges. Evaluating results of the subgroups, differences in the means of SNA, SNB and overjet between the groups were observed. Analysis of the mandibular growth pattern revealed, that neither vertical nor horizontal patterns are dominant in hypodontia patients. Conclusions In certain dentofacial parameters differences between persons with hypodontia and such with full dentition exist. According to our findings agenetic teeth may have a negative influence on the saggital development of a jaw and the lower face and may be responsible for increased overbites. This should receive attention in orthodontic treatment of hypodontia patients

Healthy ageing of cloned sheep

The health of cloned animals generated by somatic-cell nuclear transfer (SCNT) has been of concern since its inception; however, there are no detailed assessments of late-onset, non-communicable diseases. Here we report that SCNT has no obvious detrimental longterm health effects in a cohort of 13 cloned sheep. We perform musculoskeletal assessments, metabolic tests and blood pressure measurements in 13 aged (7–9 years old) cloned sheep, including four derived from the cell line that gave rise to Dolly. We also perform radiological examinations of all main joints, including the knees, the joint most affected by osteoarthritis in Dolly, and compare all health parameters to groups of 5- and 6-year-old sheep, and published reference ranges. Despite their advanced age, these clones are euglycaemic, insulin sensitive and normotensive. Importantly, we observe no clinical signs of degenerative joint disease apart from mild, or in one case moderate, osteoarthritis in some animals. Our study is the first to assess the long-term health outcomes of SCNT in large animals