I have two cell lines, one is the wild-type HeLa Cell Line (wt-HeLa), and another is a virally transfected with Connexin-43 HeLa Cell Line (HeLa-43). I have grown both the cultures overnight, keeping the number of seeded cells equal. Next Day, I observed my cells. wt-HeLa had grown to 80% confluency as usual. HeLa-43 was at 40% confluency, but the cells grew in aggregates of 8-10 cells. When I let HeLa-43 grow further, I observed colonies of cells, but much later. What can be the possible explanation for this ??
How is the expression of Connexin regulated? As you say, it's virally transfected, I assume also a viral promoter? And as you overexpress a gap-junction protein, the cell aggregation could be a result of more gap junctions, and thereby, more stable cell-to-cell interactions.
Thanks for the answer. Sure, the cell has a viral promoter. But the reason I transfected wt-HeLa cells with Cx-43 cDNA because wt-HeLa lacks Connexins/present in negligible amounts. And Yes, as I increased levels of Cx-43, Gap Junction Intercellular Communication (GJIC) increased, as a result, anchorage of one cell to another cell also increased. Hence, the culture grows in aggregations/colonies.
But, my main question was why I observed a much slower rate of growth for the transfected cells. I suspect that, due to abnormally high Cx-43, the localization of Cx-43 is much different than any other Connexin positive cell line. Hence, it can be expected that the maintenance of Homeostasis would require a greater amount of resources and energy, making the growth rate much slower than a typical Connexin positive Cell Line.
As far as i known, slower grow rate is generally observed, when overexpressing proteins, as a significant amount of resources are used to express your protein(s).
To overcome this, you could use a inducable promoter. Then the cells should behave like wt cells. And after gaining biomaterial, you could start you expression by activating your promoter. But I don't know, if this is doable in your experimental workflow
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