J.W. Carnwath

Cross-species hybridisation of human and bovine orthologous genes on high density cDNA microarrays

BackgroundCross-species gene-expression comparison is a powerful tool for the discovery of evolutionarily conserved mechanisms and pathways of expression control. The usefulness of cDNA microarrays in this context is that broad areas of homology are compared and hybridization probes are sufficiently large that small inter-species differences in nucleotide sequence would not affect the analytical results. This comparative genomics approach would allow a common set of genes within a specific developmental, metabolic, or disease-related gene pathway to be evaluated in experimental models of human diseases. The objective of this study was to investigate the feasibility and reproducibility of cross-species analysis employing a human cDNA microarray as probe.ResultsAs a proof of principle, total RNA derived from human and bovine fetal brains was used as a source of labelled targets for hybridisation onto a human cDNA microarray composed of 349 characterised genes. Each gene was spotted 20 times representing 6,980 data points thus enabling highly reproducible spot quantification. Employing high stringency hybridisation and washing conditions, followed by data analysis, revealed slight differences in the expression levels and reproducibility of the signals between the two species. We also assigned each of the genes into three expression level categories- i.e. high, medium and low. The correlation co-efficient of cross hybridisation between the orthologous genes was 0.94. Verification of the array data by semi-quantitative RT-PCR using common primer sequences enabled co-amplification of both human and bovine transcripts. Finally, we were able to assign gene names to previously uncharacterised bovine ESTs.ConclusionsResults of our study demonstrate the harnessing and utilisation power of comparative genomics and prove the feasibility of using human microarrays to facilitate the identification of co-expressed orthologous genes in common tissues derived from different species.

Comparison of real-time polymerase chain reaction and end-point polymerase chain reaction for the analysis of gene expression in preimplantation embryos.

The aim of the present study was to compare real-time polymerase chain reaction (PCR) and end-point PCR with respect to their suitability for the analysis of gene expression in samples in which the number of cells is limited; for example, in studies of preimplantation embryonic development and to determine the variability of the real-time reverse transcription-PCR assay. The sensitivity, dynamic range and precision of both PCR systems were compared using a single mouse liver cDNA standard. The real-time system was 100-fold more sensitive than the end-point system and had a dynamic range of more than four orders of magnitude. The linear range for end-point PCR extended for two orders of magnitude using a fixed end-point of 31 cycles. The percentage standard error of the mean based on 30 replicates was 0.14% of the threshold cycle (Ct) value for the real-time system and 6.8% for the end-point fluorescence intensity. The coefficients of variation (CV) for reverse transcription combined with real-time analysis and the complete gene expression protocol consisting of mRNA isolation, reverse transcription and real-time PCR analysis were 0.6% and 1.4% of the Ct values, respectively. The present paper details, for the first time, measurement of the biological variation of individual mammalian oocytes. The CV was 1.8% of the Ct value for expression analysis of six bovine oocytes. The results are discussed in relation to the analysis of gene expression in preimplantation embryo development.

Strategies to express factor VIII gene constructs in the ovine mammary gland

Abstract The transgenic technology focused on the production of recombinant proteins of therapeutic value in the milk of mammals has been increasingly successful in recent years. We have approached the problem of expressing human coagulation factor VIII in the lactating mammary gland of sheep by employing the whey acidic protein and the β-lactoglobulin promoter elements to drive transcription of factor VIII cDNA in appropriate gene constructs. To understand and eventually optimize expression of the factor VIII cDNA in transgenic animals, several classes of constructs were produced, in which expression is controlled by either the well characterized murine metallothionein I promoter or by one of the two mammary gland-specific promoter elements. In attempts to increase the efficiency of factor VIII production in transgenic animals, factor VIII cDNA-containing constructs were produced that include the introns of the metallothionein gene and/or that have had the sequences encoding the B-domain of factor VIII deleted. Transgenic mice and sheep have been obtained by microinjection of some of these constructs into zygotes and factor VIII gene expression in lactating animals is under investigation.

Expression of the gap junction gene Connexin43 (CX43) in preimplantation bovine embryos derived in vivo or in vitro

In this study we have examined the presence of mRNA encoding connexin 43 (Cx43) in bovine embryos derived in vivo and in vitro. Cumulusp=n-oocytecomplexes, immature and matured oocytes liberated from cumulus cells, zygotes, 2p=n-4-celland 8p=n-16-cellembryos, morulae, blastocysts and hatched blastocysts were produced in vitro from ovaries obtained from an abattoir using TCM 199 supplemented with hormones and 10% oestrous cow serum for maturation. Cumulusp=n-oocytecomplexes matured for 24 h were exposed to bull spermatozoa for 19 h and then cultured in TCM 199 supplemented with 10% oestrous cow serum to the desired developmental stage. Morulae and blastocysts derived in vivo were collected from superovulated donor cows. Total RNA was extracted from pools of 60p=n-200 bovine oocytes or embryos using a modified phenolp=n-chloroformextraction method and analysed by reverse transcriptase polymerase chain reaction. Before reverse transcription, aliquots of DNase-digested embryonic RNA were tested by polymerase chain reaction using bovine-specific primers to control for residual genomic DNA contamination. DNAx=req- free, total RNA was reverse transcribed after preincubation with the Cx43 specific 3m=primer. The resultant cDNA was amplified by polymerase chain reaction using Cx43 specific primers that define a 516 bp fragment of Cx43. The reverse transcriptase polymerase chain reaction product was verified by restriction enzyme analysis with Alu I and sequencing. Assays were repeated at least twice for each developmental stage and provided identical results between replicates. Cx43 transcripts were detected in bovine morulae and blastocysts grown in vivo. In contrast, whereas the early in vitro stages from cumulusp=n-oocytecomplexes to morulae expressed Cx43, blastocysts and hatched blastocysts did not have detectable concentrations of mRNA from this gene. Restriction enzyme cutting revealed three fragments of the predicted size (139, 177, 200 bp). The amplified product showed 100% identity with the published bovine genomic DNA sequence. Under our in vitro conditions the Cx43 gene either had never been activated, which would require that the maternal transcript was stable through early development, or embryonic gene expression that had been active was then terminated prematurely. The differences in transcription between bovine embryos derived in vivo or in vitro indicate that culture conditions affect gene expression. This affords a tool for the further optimization of in vitro production systems for bovine embryos and contributes towards physiological characterization by definition of transcription phenotype of bovine embryos produced in vitro.

DNA-Fingerprintuntersuchungen bei 4 Ziegenrassen zur Auswertung populationsgenetischer Parameter n einschließlich bei der gefährdeten Thüringer Wald Ziege

Abstract. Title ofthe paper: Investigations of population genetic parameters using multilocus DNA fingerprinting in 4 goat breeds in Germany Conventional fmgerprint banding pattems were produced by Southern blotting and digoxigenm labeled oligonucleotid probes (GTG)5 and (GT) g . Digitised images of the blots were analysed with the RFLPscan Computer program. These probes produced more bands (20–39 distinct informative bands within each group) in these goat breeds than in sheep breeds. Typically, in individual goats, an average of 8–11 bands were detected in the range of 4.3–23 kb. Analysis ofthe Polymorphie banding pattems showed that 4.5 to 8.3 genetic loci were represented by these probes. The analysis of pooled DNA samples (10–28 animals per lane) overestimated similarity values (S) between different breeds. Average similarity values within groups (based on RFLPs of individuals) range from 0.42–0.61, while similarity values between different groups range from 0.36–0.47. The results indicated that native Thuringer Wald Ziege are different from the Toggenburger goats.The Heterozygosity index ( H ) for each breed calculated from (GTG)5 fingerprints ( H = 0.42–0.62) were in good agreement (negative correlated) with band sharing valve. These H values are lower than those obtained in a similar study of 19 sheep breeds in which the range was (0.68–0.82). The goat breed with the highest H value was the Bunte Deutsche Edelziege (H = 0,62). Ranking the breeds by heterozygosity index and by inversely band sharing index gives: BDE > German TOGO > WDE, TWZ > Swiss TOGO. The results are discussed with respect to breeding programs and the influence of small population size.