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Biomedical subjects

M D Bishop

Publications and source records attributed to M D Bishop.

At least 19 recordsLinked to original sources
PubMed2001

Validation of a reverse transcription nested polymerase chain reaction (RT-nPCR) to detect bovine viral diarrhea virus (BVDV) associated with in vitro-derived bovine embryos and co-cultured cells.

Sensitive RT-nPCR assays can be used for the rapid detection of viruses. The objective of this research was to validate an RT-nPCR assay for detection of BVDV associated with various samples collected from an IVF system. In 12 research replicates, we maintained matured COCs as negative controls or exposed them to 1 of 4 noncytopathic strains (SD-1, NY-1, CD-87, or PA-131) of BVDV for 1 h immediately before IVF. After 4 d of IVC, we harvested groups of 5 nonfertile ova or degenerated embryos (NFD) and some associated cumulus cells and transferred developing embryos and the remaining cumulus cells into secondary IVC drops. On the seventh d of IVC, cumulus cells, groups of 5 washed NFD and groups of 5 developed, washed embryos were harvested. We also collected single developed embryos after washing, washing with trypsin, washing and cryopreservation in ethylene glycol, or washing with trypsin and cryopreservation in ethylene glycol. All washes were performed according to International Embryo Transfer Society standards. Developed embryos and NFD were sonicated prior to assay. All samples were assayed for BVDV using virus isolation and RT-nPCR. The virus isolation and RT-nPCR assays determined that all negative control samples were BVDV-free. Virus was detected in association with all exposed cumulus cells and groups of developed embryos using both virus isolation and RT-nPCR. Results from viral assays of other exposed samples indicate enhanced sensitivity of the RT-nPCR assay. The RT-nPCR assay used in this research exhibited acceptable sensitivity, specificity, predictive value and repeatability for rapid detection of BVDV associated with the various samples obtained from an IVF system.

Animals↗
PubMed2000

Developing efficient strategies for the generation of transgenic cattle which produce biopharmaceuticals in milk.

At the close of the millennium, a revolution in the treatment of disease is taking shape due to the emergence of new therapies based on human recombinant proteins. The ever-growing demand for such pharmaceutical proteins is an important driving force for the development of safe and large-scale production platforms. Since the efficacy of a human protein is generally dependent on both its amino acid composition as well as various post-translational modifications, many recombinant human proteins can only be obtained in a biologically active conformation when produced in mammalian cells. Hence, mammalian cell culture systems are often used for expression. However, this approach is generally known for limited production capacity and high costs. In contrast, the production of (human) recombinant proteins in milk of transgenic farm animals, particularly cattle, presents a safe alternative without the constraint of limited protein output. Moreover, compared to cell culture, production in milk is very cost-effective. Although transgenic farm animal technology was still in its infancy a decade ago, today it is on the verge of fulfilling its potential of providing therapeutic proteins that can not be produced otherwise in sufficient quantities or at affordable cost. Since 1989, we have been at the forefront of this development, as illustrated by the birth of Herman, the first transgenic bull. In this communication, we will present an overview of approaches we have taken over the years to generate transgenic founder animals and production herds. Our initial strategies were based on microinjection; at the time the only viable option to generate transgenic cattle. Recently, we have adopted a more powerful approach founded on the application of nuclear transfer. As we will illustrate, this strategy presents a breakthrough in the overall efficiency of generating transgenic animals, product consistency, and time of product development.

Animals↗
PubMed2000

Quality controls for bovine viral diarrhea virus-free IVF embryos.

Introduction of bovine viral diarrhea virus (BVDV) with cumulus-oocyte-complexes (COCs) from the abattoir is a concern in the production of bovine embryos in vitro. Further, International Embryo Transfer Society (IETS) guidelines for washing and trypsin treatment of in-vivo-derived bovine embryos ensure freedom from a variety of pathogens, but these procedures appear to be less effective when applied to IVF embryos. In this study, COCs were exposed to virus prior to IVM, IVF and IVC. Then, virus isolations from cumulus cells and washed or trypsin-treated nonfertile and degenerated ova were evaluated as quality controls for IVF embryo production. The effect of BVDV on rates of cleavage and development was also examined. All media were analyzed prior to the study for anti-BVDV antibody. Two approximately equal groups of COCs from abattoir-origin ovaries were washed and incubated for 1 h in minimum essential medium (MEM) with 10% equine serum. One group was incubated in 10(7) cell culture infective doses (50% endpoint) of BVDV for 1 h, while the other was incubated without virus. Subsequently, the groups were processed separately with cumulus cells, which were present throughout IVM, IVF and IVC. Cleavage was evaluated at 4 d and development to morulae and blastocysts at 7 d of IVC. After IVC, groups of nonfertile and degenerated ova or morulae and blastocysts were washed or trypsin-treated, sonicated and assayed for virus. Cumulus cells collected at 4 and 7 d were also assayed for virus. Anti-BVDV antibody was found in serum used in IVM and IVC but not in other media. A total of 1,656 unexposed COCs was used to produce 1,284 cleaved embryos (78%), 960 embryos > or = 5 cells (58%), and 194 morulae and blastocysts (12%). A total of 1,820 virus-exposed COCs was used to produce 1,350 cleaved embryos (74%), 987 embryos > or = 5 cells (54%), and 161 morulae and blastocysts (9%). Rates of cleavage (P = 0.021), cleavage to > or = 5 cells (P = 0.026) and development to morula and blastocyst (P = 0.005) were lower in the virus-exposed group (Chi-square test for heterogeneity). No virus was isolated from any samples from the unexposed group. For the exposed group, virus was always isolated from 4- and 7-d cumulus cells, from all washed nonfertile and degenerated ova (n = 40) and morulae and blastocysts (n = 57) and from all trypsin-treated nonfertile and degenerated ova (n = 80) and morulae and blastocysts (n = 91). Thus, virus persisted in the system despite the presence of neutralizing antibody in IVM and IVC media, and both washing and trypsin treatment were ineffective for removal of the virus. Presence of virus in 4- and 7-d cumulus cells as well as in nonfertile and degenerated ova were good indicators of virus being associated with morulae and blastocysts.

Animals↗
PubMed2000

Differential control during maximal concentric and eccentric loading revealed by characteristics of the electromyogram.

Maximal eccentric loading has been associated with higher levels of spindle afferent activity but lower levels of integrated EMG as compared to maximal concentric loading. Elbow flexor EMG was recorded from 17 subjects during concentric (CONC) and eccentric (ECC) elbow flexion at 70 degrees s(-1) using a Kin-Com dynamometer. We hypothesized that peak EMG amplitude would be more sensitive to fluctuations in facilitation by the spindle primary afferents via the segmental stretch reflex pathway, and that the mean EMG would be more reflective of the ongoing level of muscle activation. A ratio of peak to mean EMG (P/M EMG ratio) was predicted to be larger during maximal eccentric loading than maximal concentric loading. The peak EMG (P<0.013) and the P/M EMG ratio (P<0.001) were significantly greater during the ECC condition than the CONC condition. In a subgroup of three subjects who underwent 3 weeks of eccentrically biased weight training, EMG, peak torque and torque variability were assessed before and after training. P/M EMG ratio decreased, while peak torque and torque variability increased following the training. Differences in the P/M EMG ratio appear to reflect differences in the way eccentric and concentric muscle actions are controlled and do not simply represent less control during the eccentric task.

Adult↗
PubMed1999

Physical assignment of the bovine MHC class IIa and class IIb genes.

Screening of a bovine yeast artificial chromosome (YAC) library revealed two clones which contain most of the class II genes of the major histocompatibility complex (MHC) known to date. The YACs were mapped by fluorescence in situ hybridization (FISH) and characterized for the class II genes they contain. We found that the classic class II genes BoLA- DQA, -DQB, -DRA, and -DRB3 are located at BTA 23q21 and the non-classic class II genes DYA, DIB, LMP2, LMP7, TAP2, BoLA-DOB, -DMA, -DMB, and -DNA are located at BTA 23q12-->q13. These two different mapping locations confirm and extend previous findings of a gross physical distance between classic and non-classic MHC class II genes in cattle.

Animals↗
PubMed1998

Mapping quantitative trait loci for milk production and health of dairy cattle in a large outbred pedigree.

Quantitative trait loci (QTL) affecting milk production and health of dairy cattle were mapped in a very large Holstein granddaughter design. The analysis included 1794 sons of 14 sires and 206 genetic markers distributed across all 29 autosomes and flanking an estimated 2497 autosomal cM using Kosambi's mapping function. All families were analyzed jointly with least-squares (LS) and variance components (VC) methods. A total of 6 QTL exceeding approximate experiment-wise significance thresholds, 24 QTL exceeding suggestive thresholds, and 34 QTL exceeding chromosome-wise thresholds were identified. Significance thresholds were determined via data permutation (for LS analysis) and chi-square distribution (for VC analysis). The average bootstrap confidence interval for the experiment-wise significant QTL was 48 cM. Some chromosomes harbored QTL affecting several traits, and these were always in coupling phase, defined by consistency with genetic correlations among traits. Chromosome 17 likely harbors 2 QTL affecting milk yield, and some other chromosomes showed some evidence for 2 linked QTL affecting the same trait. In each of these cases, the 2 QTL were in repulsion phase in those families appearing to be heterozygous for both QTL, a finding which supports the build-up of linkage disequilibrium due to selection.

Analysis of Variance↗
PubMed1998

Report of the first workshop on the genetic map of bovine chromosome 1.

A report of the first workshop on the genetic map of bovine chromosome 1 (BTA1) is presented. Five laboratories contributed 31,962 informative meioses from 70 loci. Thirty-two loci which had been typed by at least two laboratories were used to construct a framework genetic map with a likelihood ratio support of at least 1000:1 for locus order. The resulting sex-averaged framework map contained 26 loci and spanned 163.6 CM. The lengths of the female and male maps were 159.5 CM and 165.3 CM, respectively, and there was evidence for an expansion in the telomeric one-third of the male map. Of the four cases where order for closely linked loci differed among the maps produced for each of the contributing laboratories, a consensus order was obtained for three in the framework map. The average genetic distance between framework loci on the sex-averaged map was 6.3 CM.

Animals↗
PubMed1997

Comparative linkage mapping of human chromosome 13 and bovine chromosome 12.

A comparative linkage map of human chromosome 13 and bovine chromosome 12 was constructed using eight polymorphic microsatellite markers associated with six specific genes. Linkage of these was also examined relative to five previously mapped anonymous microsatellite markers. Seven gene-linked markers were developed from bovine large-insert genomic clones containing one of five genes of interest (serotonin receptor subtype 2, fms-related tyrosine kinase, coagulation factor 10, retinoblastoma susceptibility gene, collagen type IV alpha 1), and one additional marker was developed from a microsatellite resident within an intron of the bovine dopachrome tautomerase gene. Four of these loci were previously assigned to bovine chromosome 12 by analysis of a somatic cell hybrid panel. This study provides linkage information for examining gene order in this conserved synteny group. The comparative linkage mapping results indicate that the q arm of human chromosome 13 is almost entirely conserved in bovine chromosome 12. One intrachromosomal rearrangement was detected in this linkage group relative to human, and this rearrangement was confirmed by fluorescence in situ hybridization results.

Animals↗
PubMed1997

A medium-density genetic linkage map of the bovine genome.

A cattle genetic linkage map was constructed which covers more than 95 percent of the bovine genome at medium density. Seven hundred and forty six DNA polymorphisms were genotyped in cattle families which comprise 347 individuals in full sibling pedigrees. Seven hundred and three of the loci are linked to at least one other locus. All linkage groups are assigned to chromosomes, and all are orientated with regards to the centromere. There is little overall difference in the lengths of the bull and cow linkage maps although there are individual differences between maps of chromosomes. One hundred and sixty polymorphisms are in or near genes, and the resultant genome-wide comparative analyses indicate that while there is greater conservation of synteny between cattle and humans compared with mice, the conservation of gene order between cattle and humans is much less than would be expected from the conservation of synteny. This map provides a basis for high-resolution mapping of the bovine genome with physical resources such as Yeast and Bacterial Artificial Chromosomes as well as providing the underpinning for the interpolation of information from the Human Genome Project.

Animals↗
PubMed1997

Isolation of microsatellites from a bovine YAC clone harbouring the SOD1 gene.

A bovine yeast artificial chromosome (YAC) clone containing the superoxide dismutase 1 (SOD1) gene was used as a template for polymerase chain reaction (PCR) amplification using a conserved short interspersed nuclear element (SINE) primer. Two highly polymorphic microsatellites with nine and eight alleles were isolated and mapped by linkage analysis to the centromeric region of BTA1. These microsatellites will be used in the construction of a genetic and physical map of the SOD1 region towards positional cloning of the polled gene.

Animals↗