Our results resemble those reported initially when using the bacterial b-gal gene as a reporter in the context of the VavP promoter that was expressed in a variegated manner and subjected to frequent silencing over time. PEV is a well known phenomenon in transgenesis depending on chromatin structures near the site of insertion, most pronounced when transgene constructs are used that lack a defined locus control region, but also hard to distinguish in our case from gene silencing-induced mosaicism. Based on experiences using hCD4 or hBcl-2 as transgenes in the VavP vector showing uniform expression of the human transgenes tested, we can only speculate that the non-mammalian Venus sequence, although codon optimized for the use in mammalian cells, may be 5α-Androstane-3α,17β-diol susceptible to stochastic epigenetic silencing in a certain percentage of cells, as was observed previously when VavP-lacZ tg mice were compared to VavP-hCD4 tg mice. Alternatively, the pOPI3-CAT derived SV40-derived intronic sequence containing the lacO sites may be subjected to epigenetic silencing, as noted before when bacterial or viral sequences were introduced into mammalian genome. However, the latter seems unlikely, since we noted similar patterns of transgene expression in the VV strain, lacking this DNA element. Since we were forced to use different restriction enzymes to linearize the modified VLV plasmid, it remains also possible that the residual bacterial DNA sequence of about 250 base pairs that we could no longer remove may render the transgene more susceptible to silencing. Alternatively, high-copy BMS-214662 hydrochloride number insertion of transgenes reportedly favours variegation of expression due to repeat-induced gene silencing which may in part explain why VV mice, that do have a higher copy number insertion than VLV mice based on qPCR analysis, showed stronger variegation. Alternatively, high-level expression of Venus may not be well tolerated in haematopoietic cells causing silencing of transgene expression over time or counter selection in stem cells. High-level GFP expression was reported to induce apoptosis in cultured cells and cardiomyopathy was observed in FVB mice with high-level expression of GFP when driven from the cardiac alpha-myosin heavy chain promoter. However, our in vitro analysis did not show increased cell death in Venus expressing lymphocytes and counter selection due to toxicity in leukocytes can be largely excluded since we did not see a significant reduction of the percentage of Venus + cells in peripheral blood over time.