Relative transgene expression frequencies in homozygous versus hemizygous transgenic mice.

We have used a simple binomial model of stochastic transgene inactivation at the level of the chromosome or transgene, rather than the cellular level, for the analysis of two mouse transgenic lines that show variegated patterns of expression. This predicts the percentages of cells that express one, both or neither alleles of the transgene in homozygotes from the observed percentages of cells, which express the transgene in hemizygotes. It adequately explained the relationship between the numbers of cells expressing the transgene in hemizygous and homozygous mosaic 21OH/LacZ mouse adrenals and mosaic BLG/7 mouse mammary glands. The binomial model also predicted that a small proportion of cells in mosaic mammary glands of BLG/7 homozygotes would express both BLG/7 alleles but published data indicated that all cells expressing the transgene showed monoallelic expression. Although it didn't fit all of the BLG/7 data as precisely as a more complex model, which used several ad hoc assumptions to explain these results, the simple binomial model was able to explain the relationship in observed transgene expression frequencies between hemizygous and homozygous mosaic tissues for both 21OH/LacZ and BLG/7 mice. It may prove to be a useful general model for analysing other transgenic animals showing mosaic transgene expression.

An attempt to investigate the presence of Epstein Barr virus in multiple sclerosis and normal control brain tissue.

Multiple Sclerosis (MS) is an important demyelinating disease of the central nervous system, the aetiology of which may possibly have a viral component at some stage. In this study we investigated the possible involvement in MS of the human herpes virus Epstein Barr Virus (EBV). Utilising both fluorescent and non-fluorescent in situ hybridisation (FISH) techniques, we examined human post mortem tissues obtained from a tissue bank for the presence of immediate early and late viral gene and protein expression in MS patient normal appearing white matter (NAWM), lesional tissue and normal control brain samples. The technique of mRNA FISH showed that many of the tissues were largely degraded and therefore could not provide any evidence of viral gene expression. Some weak scattered signals, however, were seen in mRNA ISH for both lytic and latent gene transcription in all three tissue categories. The failure of IF and mRNA FISH in the majority of samples alongside the poor signal for mRNA ISH precluded any definite conclusions to be made as to the possible ongoing involvement of EBV in MS. While certainly not ruling out a possible role of EBV in MS, especially in the context of a 'hit and run' mechanism, these studies illustrate the difficulties of using autopsy tissues for molecular studies when tissue preservation is sub-optimal. Nevertheless, the limited data obtained did not provide any positive evidence of EBV involvement.

Investigating the presence of human herpesvirus 7 and 8 in multiple sclerosis and normal control brain tissue.

Multiple sclerosis (MS) is an important demyelinating disease of the central nervous system, the aetiology of which is thought to have a possible viral component. In this study we investigated the possible involvement in MS of two herpes viruses: the neurotropic human herpesvirus 7 (HHV-7) and the related human herpesvirus 8 (HHV-8). Utilising fluorescent in situ hybridisation (FISH) techniques, we examined human post mortem tissues for the presence of immediate early and late viral gene or protein expression in MS patient normal appearing white matter (NAWM), lesional tissue and normal control brain samples. HHV-7 and/or HHV-8 mRNA or protein was detected in some individuals in all three sample categories and was restricted to oligodendrocytes, as determined by double mRNA FISH analysis or immuno fluorescence (IF). No samples showed evidence of viral mRNA when subjected to RT-PCR on extracted ribonucleic acid. We therefore conclude that there is little evidence in our particular sample cohort to suggest involvement of either HHV-7 or HHV-8 in MS.

Early and late HHV-6 gene transcripts in multiple sclerosis lesions and normal appearing white matter.

Multiple sclerosis is an inflammatory demyelinating disease of the CNS, the aetiology of which is believed to have both genetic and environmental components. We have investigated one of the candidate viruses for the environmental component of multiple sclerosis, the neurotropic human herpesvirus 6 (HHV-6). Utilizing fluorescent in situ hybridization (FISH) techniques, we have examined human post-mortem tissues for the presence of immediate early and late viral gene expression in multiple sclerosis patient normal appearing white matter (NAWM), lesional tissue and normal control brain samples. HHV-6 gene transcription was detected in all tissue samples and was restricted to oligodendrocytes, as determined by double mRNA FISH analysis. Quantitative analysis of viral mRNA expression indicated that both NAWM and lesional multiple sclerosis samples exhibited significantly higher levels of HHV-6 expression compared with the normal control samples. Lesional samples exhibited the highest levels of viral gene expression, with NAWM exhibiting an intermediate level between lesional and control tissues. Immunofluorescence against early and late HHV-6 proteins verified active translation of HHV-6 viral mRNA in oligodendrocytes. Southern blot analysis of nested polymerase chain reactions using extracted genomic DNA and cDNA confirmed the presence of the HHV-6 genome in all individuals, with the active expression profile mirroring the FISH results. The frequent high level of HHV-6 infection in multiple sclerosis samples suggests a possible role in pathogenesis.

Mono-allelic expression of variegating transgene locus in the mouse.

We have generated transgenic mice which express an ovine beta-lactoglobulin transgene during lactation. In two transgenic lines, BLG/7 and BLG/45, beta-lactoglobulin protein levels vary between siblings, reflected at the cellular level by a mosaic transgene expression pattern in the mammary tissue that is reminiscent of position effect variegation. To investigate whether this variegating expression profile can be affected by the introduction of an identical variegating locus on the homologous chromosome, we compared the beta-lactoglobulin expression profiles in mice hemizygous or homozygous for the transgene locus. In BLG/45 mice, milk protein analysis revealed that transgene expression was effectively doubled in homozygous compared to hemizygous mice. In contrast, beta-lactoglobulin protein in hemizygous and homozygous BLG/7 mice displayed a similar range; although minimum expression levels were doubled in the homozygous population, the maximum level of expression was indistinguishable between the two populations. Fluorescent in situ hybridisation (FISH) for transgene mRNA indicated that for a given protein level, the extent of cellular expression is similar in both BLG/7 populations. In homozygous mice genomic DNA and nuclear RNA FISH demonstrated that only one of the two BLG/7 loci is active in expressing cells, while two transcription foci were present in BLG/45 homozygous mice. This mono-allelic transgene expression pattern is not inherited through the germline, as hemizygous mice bred from homozygous parents expressed at the expected hemizygous population level. We discuss these observations in the context of known epigenetic events such as imprinting and trans-inactivation.

Multiple effects of genetic background on variegated transgene expression in mice.

BLG/7 transgenic mice express an ovine beta-lactoglobulin transgene during lactation. Unusually, transgene expression levels in milk differ between siblings. This variable expression is due to variegated transgene expression in the mammary gland and is reminiscent of position-effect variegation. The BLG/7 line was created and maintained on a mixed CBA x C57BL/6 background. We have investigated the effect on transgene expression of backcrossing for 13 generations into these backgrounds. Variable transgene expression was observed in all populations examined, confirming that it is an inherent property of the transgene array at its site of integration. There were also strain-specific effects on transgene expression that appear to be independent of the inherent variegation. The transgene, compared to endogenous milk protein genes, is specifically susceptible to inbreeding depression. Outcrossing restored transgene expression levels to that of the parental population; thus suppression was not inherited. Finally, no generation-dependent decrease in mean expression levels was observed in the parental population. Thus, although the BLG/7 transgene is expressed in a variegated manner, there was no generation-associated accumulated silencing of transgene expression.