TITLE: Cell lines carrying mutations in genes of interest
CASE NUMBER: C13619
ABSTRACT
The invention is a series of cell lines. Each cell line carries mutations in a specific gene of interest. The mutant cell lines were generated by (a) transfecting a cell line with a proprietary gRNA/Cas9-3xNLS/FP/TK/puro expression plasmid designed to create double strand breaks in the gene of interest, (b) selecting for cells that had taken up and expressed the plasmid, (c) expanding single cell clones, (d) testing multiple clones for loss of expression of the protein encoded by the gene, (e) expanding the line, (f) introducing Cre recombinase into the mutant cell line to remove the introduced Cas9-3xNLS/FP/TK/puro ORF, and (g) selecting for desired cells by growth in ganciclovir.
FEATURES
1. The cell lines carry unique mutations in the gene of interest.
2. The cell lines were generated with a plasmid that has a unique Cas9-3xNLS/FP/TK/puro ORF that is flanked by loxP sites, followed by deletion of the ORF following the expression of Cre recombinase activity in the cells
3. The cells lack expression of the protein encoded by the mutant gene, as assessed by immunoblot.
DECRIPTION DETAIL
The cell lines lack expression of the protein, as assessed by immunoblot. Puro plasmid was used for making these mutant cell lines, and immunoblot data of HEK293 cells that are mutant for the genes encoding the Alix, CD9, CD81, or CD63 genes were obtained. We have also generated mutant forms of other cell lines (e.g. 293T cells, Lapc-4 cells, etc.) in other genes (e.g. PSA, Rab27a, etc.).
ASSOCIATED PUBLICATIONS: None at this time
