Fig. 2

The construction of APCs with GAL-1 gene knocked out. A T7EI digestion products from twenty single-cell colonies were analyzed by agarose gel electrophoresis. DNA fragments of the GAL-1 gene with mutated sites were indicated by red arrows (2, 6, 7, 9, 10, 12, 14, 15, 18). B Sequencing of APC colonies with GAL-1 gene mutation. There were four types of mutations: 1. 13 bp deletion; 2. 7 bp deletion; 3. Insert 3 bp, black shading as substitution base; 4. 5 bp deletion and 2 bp replacement, yellow shading as substitution base; C the expression of GAL-1 protein in four mutant cell colonies were detected by Western blotting. The GAL-1 proteins of the 1st, 2nd, and 4th mutant cell colonies were completely knocked out. D Detection of GAL-1 protein in the serum-free culture medium of cell colonies by ELISA. E The expression of GAL-1 protein in cell colonies was detected by immunofluorescence, scale bar = 200 μm. APC^WT: Wild-Type APCs; APC^Vector: APCs infected with empty vector (no gRNA sequence attached); APC^{GAL−1−/−}: APCs with GAL-1 gene knocked out