Figure 1

Proviral silencing in embryonic cells – hypothesis and experimental procedure. (A) Scheme illustrating two alternatives for the proviral escape from silencing: if the regulation of expression is fully “extrinsic”, both proviruses integrated in one cell will be expressed or silenced in a synchronous and coordinated manner. That pattern might, for example, be indicative of a distinctive cellular state of the transcriptional network. A hypothetical dot plot describing this type of population is illustrated on the left. An alternative is an intrinsic or gene-specific control of the expression. Here the two proviruses in one cell will each be controlled independently and thus can be expressed asynchronously, independently of each other. This will lead to a heterogeneous population of cells expressing either red, green, both, or no marker gene, as illustrated in the dot plot on the right. (B) Illustration of the experimental procedure: one MMLV viral vector carrying a GFP reporter and a second one carrying an mCherry reporter were each packaged into virus particles and used together to infect F9 EC cells at high MOI. The infected cells were grown for 6 days and then sorted by fluorescence-activated cell sorting (FACS) to four different populations. The populations were then grown in culture, and most centrally for this study, after three weeks the initially double positive population was sorted a second time, this time into five different populations. These cells were used in the experiments described herein.