Table 2.
Transfection conditions and targeting efficiency for loci expressed in undifferentiated hESCs.
| Targeted locus* | YearREF | Electroporation settings | No. of antibioticR colonies/starting cell no. | No. properly targeted colonies/total | Positive selection | Negative selection | Homology arms 5′/3′ (Kb) |
|---|---|---|---|---|---|---|---|
| HPRT* | 2003 8 | 220V 960 μF 320V 200 μF | ∼1/107 350 NeoR/1.5 × 107; 50/350 GancyclovirR; 7/50 6‐TGR | 7/350 (2%) 7/50 (14%) 7/7 (100%) | Neo Neo Neo Neo | TK & 6‐TG TK TK and 6‐TG | 1.9/10 |
| 2004 19 | ExGen 500 Reagent | 2/1 × 108 | 2/2 (100%) | Hygro | 6‐TG | 8.5 total | |
| 2008 22 | 300V 100μSec 320V 200μF | 488 NeoR/1 × 106 cells 233 NeoR/1 × 106 cells 600 NeoR/1 × 106 cells | 1/488 (0.2%) 1/233 (0.43%) 1/600 (0.16%) | EF1α‐Neo EF1α‐Neo EF1α‐Neo | – | 4.8/2.1 | |
| 2010 24 | 250V 4 ms × 2 | 424 NeoR/2–4 × 106 cells | 6/424 (1.4%) | PGK‐Neo | – | 7/2 | |
| 2010 14 | 320V 200μF | Data not provided | Data not provided | CAG‐Neo‐IRES‐Puro | – | 118/77 | |
| OCT4 | 2003 8 | 320V 200μF | 103 NeoR/1.5 × 107 cells | 28/103 (27%) 22/56 (40%) | Neo | – | 6.3/1.6 6.3/6.5 |
| ROSA26 | 2007 23 | 250V 500μF | 24/5 × 106 cells 64/5 × 106 cells | 1/24 (4.1%) 1/64 (1.5) Avg: 2/88 (2.3%) | Neo | DTA | 1.0/4.1 |
| ATM | 2010 14 | 320V 200μF | 10–50 NeoR/2 × 107 cells | <0.5% 3/14(21%); 10/37 (27%) 2/10 (20%) with second allele | CAG‐Neo‐CAG‐Neo‐IRES‐ Puro | – | 9.0/2.0 103/6.5 |
| P53 | 2010 14 | 320V 200μF | ∼30 NeoR/2 × 107 cells (done twice; yielded 60 NeoR colonies) | 2/60 (3.3%) 7/32 (22%) | CAG‐Neo & CAG‐Neo‐IRES‐Puro | – | 77/5.5 80/81 |
| BAF250a | 2010 15 | 320V 200μF | 22 NeoR/1 × 107 cells | 16/22 (73%) | β‐geo | – | 3.9/3.7 |
| NANOG | 2010 17 | 250V 500μF or 800V 10μF | 637 Neo/1.9 × 107 cells 167 NeoR/0.9 × 107 cells | 4/637 (0.6%) HUES‐1 19/40 (11.4%) HUES‐3 | SV40‐Neo | – | 12.5/3.5 |
*See Table 1 .