I have been introducing missense mutations in several human osteoblastic lines by transfecting vectors by the double nick method, ssODN and GFP reporter of specific crispr activity. I can see GFP positive cells and confirm the mutation in pool of cells by sequencing but when I try to isolate a single cell colony they dont survive. I have tried cell sorting by GFP and manual isolation under the microscope. I also tried to conditioned media and isolation of small group of cells without result. Any suggestion about how to overcome this problem?
The cells have somehow gone quietscent or senescent after your transfection.
Which gene did you try to insert GPF into? Some tumor cells are addicted to certain oncogenes, once you disrupt them they fail to grow.
(Its interesting, you say you can insert GFP using ssODN? but I thought GFP is too big for ssODN HDR,you need dsDNA.)
Ivan,
Are your single clones failing to propagate when you first isolate them (from single cell)? Or, are you letting them expand into a multi-cell colonies and then they subsequently fail to grow? Sometimes it is necessary to isolate hundreds of single cell clones (in 96-well plates) in order to get a good number of colonies to work with. It can take as much as a week or two to get the single cell clones to expand.
- Badges
- Science method