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Genes transfected into embryonal carcinoma stem cells are both lost and inactivated at high frequency
P K Schmidt-Kastner1, K Jardine, M Cormier
1Ottawa Regional Cancer Center, Department of Medicine, Ontario, Canada.
Abstract:
Embryonal carcinoma (EC) cells can be efficiently transfected with cloned DNAs but there is a strong tendency for expression from transfected genes to be lost from stably transformed cells. To investigate the mechanism responsible for this loss of expression, we transfected P19 EC cells with a gene encoding the E. coli beta-galactosidase and examined expression of this gene in clonal populations of cells. Cells that carry and express the beta-galactosidase gene give rise to cells that do not express at a rate of about 0.02 events per cell per cell division. These non-expressing cells were of two types, some had lost the transfected genes while others had inactivated them. In those cells that retained but inactivated the transfected genes, the inactive state was stable and suppression was at the level of transcription initiation but not associated with increased DNA methylation. Because transfected DNAs integrate into the genome as tandem arrays, the gene loss and inactivation seen in EC cells may be analogous to the repeat-induced gene inactivation seen in lower eukaryotes.
Insights
Embryonal carcinoma (EC) cells often lose expression of transfected genes. This study found gene loss or stable inactivation at the transcriptional level, similar to repeat-induced gene silencing in other eukaryotes.
Area of Science:
- Molecular Biology
- Developmental Biology
- Genetics
Background:
- Embryonal carcinoma (EC) cells are amenable to DNA transfection.
- Stable expression of transfected genes in EC cells is frequently lost over time.
- Understanding gene silencing mechanisms in EC cells is crucial for genetic manipulation.
Purpose of the Study:
- To investigate the mechanisms underlying the loss of expression from transfected genes in P19 EC cells.
- To characterize the fate of transfected DNA in stably transformed cell populations.
- To determine if gene inactivation is associated with epigenetic modifications.
Main Methods:
- Transfection of P19 EC cells with the E. coli beta-galactosidase gene.
- Analysis of gene expression in clonal cell populations over time.
- Distinguishing between gene loss and gene inactivation.
- Assessment of DNA methylation and transcriptional activity.
Main Results:
- A significant rate of non-expressing cells (0.02 events/cell/division) arose from expressing cells.
- Non-expressing cells resulted from either complete gene loss or stable gene inactivation.
- Inactivated genes were suppressed at the transcriptional initiation level without increased DNA methylation.
- Transfected DNA integration as tandem arrays was observed.
Conclusions:
- Gene expression loss in EC cells involves both gene deletion and stable transcriptional silencing.
- The observed gene inactivation mechanism is independent of DNA methylation changes.
- The phenomenon may resemble repeat-induced gene silencing in lower eukaryotes.
- Findings provide insights into genetic instability in EC cells.