Repeat-induced gene silencing in mammals

Abstract

In both plants and Drosophila melanogaster, expression from a transgenic locus may be silenced when repeated transgene copies are arranged as a concatameric array. This repeat-induced gene silencing is frequently manifested as a decrease in the proportion of cells that express the transgene, resulting in a variegated pattern of expression. There is also some indication that, in transgenic mammals, the number of transgene copies within an array can exert a repressive influence on expression, with several mouse studies reporting a decrease in the level of expression per copy as copy number increases. However, because these studies compare different sites of transgene integration as well as arrays with different numbers of copies, the expression levels observed may be subject to varying position effects as well as the influence of the multicopy array. Here we describe use of the lox/Cre system of site-specific recombination to generate transgenic mouse lines in which different numbers of a transgene are present at the same chromosomal location, thereby eliminating the contribution of position effects and allowing analysis of the effect of copy number alone on transgene silencing. Reduction in copy number results in a marked increase in expression of the transgene and is accompanied by decreased chromatin compaction and decreased methylation at the transgene locus. These findings establish that the presence of multiple homologous copies of a transgene within a concatameric array can have a repressive effect upon gene expression in mammalian systems.

Evaluation of Tet-on system to avoid transgene down-regulation in ex vivo gene transfer to the CNS

“…Leakiness of the Tet system is an often-reported phenomenon that could limit its use in certain applications. 12,19,20 Several factors could contribute to the residual activity: intrinsic activity of the minimal promoter, number of copies integrated, 21 low affinity binding of rtTA to the response element despite the absence of doxycyclin, or, enhancer elements in proximity to the integration site promoting the activity of the minimal promoter contained in the TRE. 7 Regarding the last point, Bujard et al 22 and Yin et al, 20 have recommended not co-transfecting the (CMVϪ) expression unit of the transactivator and the response element since the minimal promoter might function as an enhancer trap amenable to influence by a close-by integrated CMV enhancer/promoter.…”

“…The actual mechanism associated with transgene silencing are not fully understood, however, in vitro and in vivo studies have shown that de novo methylation of transcriptionally regulating sequences or coding sequences, 30,31 acetylation of histones, 32 position effects, transgene copy number 21 and extrusion of introduced transgenes 33 are contributing factors. Including so-called insulator sequences to flank the transcriptional unit may be a promising strategy to shield the transgene from cisacting elements 34 and increase the possibility that an integrated transgene will be expressed.…”

A transgenic mouse Class‐III β tubulin reporter using yellow fluorescent protein

“…This has been discussed by Feng et al to account for the rather extreme variability in reporting Thy1 expression in twenty-five transgenic lines (Feng et al, 2000). Transgene expression can also be affected by the number of copies of the transgene that have become integrated, the site of chromosomal integration or other factors such as position effect variegation (see: Dorer and Henikoff, 1997;Garrick et al, 1998;Robertson et al, 1995;Sabl and Henikoff, 1996) . The difference in expression is not accompanied by any aberration in development as both transgenic lines develop normally and no YFP-induced toxicities are found.…”

Incomplete penetrance and variable expressivity: is there a microRNA connection?

“…This discordance is correlated with copy number, the status of DNA, and histone methylation at the site-of-integration of the transgene. (23,24) These examples reiterate that in spite of diverse mechanisms, the reinforcement of differential expression appears to be principally brought about either by DNA methylation and/or histone modifications.…”