Human and mouse embryonal carcinoma cells and early embryo cells display a unique phenotype: they do not express certain viral and cellular genes that are expressed in differentiated successors. Therefore, they provide a system to study interesting aspects of the control of gene expression during very early embryonic development. We have found evidence that primary control of gene expression in the EC cell occurs by mechanisms other than de novo methylation. Although de novo methylation occurs, it may be a consequence of nonexpression. We will examine the effects of introducing CAD or other genes into embryonal carcinoma cells to determine whether they are expressed at any time or all times after introduction to the cells. The sequences will be transformed into the cells by calcium phosphate coprecipitation or protoplast fusion. Does the expression of introduced sequences change with integration into the genome of the mouse cell? Does the expression correlate with methylation patterns of the sequences? It is becoming apparent from our work, as well as that of others, that the promoter activity of retrovirus may be specifically repressed in EC and early mouse embryo cells, perhaps by a transacting repressor-type function. However, it is possible to get expression of genes that are placed between two LTRs as long as they contain their own EC-active enhancer/promoter region. This enhancer/promotor can be anything that is active in EC cells, such as the mutant polyoma enhancer sequences, SV40 viral enhancer/promoter sequences or perhaps even a mutant py/MLV LTR U?3? region could be active if placed between two LTRs in a retrovirus vector. We plan to test this hypothesis by constructing such a retrovirus vector containing a third LTR-driven CAT or neo gene. Based on the results of the above studies we will determine the best internal promoter to be used for making a retrovirus expression vector that will be active in EC and early embryo cells. (M)
| Levy, D E; McKinnon, R D; Brolaski, M N et al. (1987) The 3' long terminal repeat of a transcribed yet defective endogenous retroviral sequence is a competent promoter of transcription. J Virol 61:1261-5 |
