1. Establishment of ES cells
(1) TT2 ES Cells from an F1 embryo between C57BL/6 and CBA
A novel ES cell line, TT2, with high germline-differentiating potency. Yagi T., Tokunaga T., Furuta Y., Nada S., Yoshida M., Tsukada T., Saga Y., Takeda N., Ikawa Y. and Aizawa S. Anal Biochem. 1993 Oct;214(1):70-6

(2) HK3i ES cells from C57BL/6N embryos
Three Inhibitors of FGF Receptor, ERK and GSK3 Establishes Germline-Competent Embryonic Stem Cells of C57BL/6N Mouse Strain with High Efficiency and Stability. Kiyonari H, Kaneko M, Abe S and Aizawa S. Genesis 2010 May;48(5):317-27.  

2. Construction of Targeting Vector
(1) long PCR Method
ang is a novel gene expressed in early neuroectoderm, but its null mutant exhibits no obvious phenotype. Murata T, Furushima K, Hirano M, Kiyonari H, Nakamura M, Suda S, Aizawa S. Gene Expr Patterns. 2004 dec;5(2):171-8

(2) BAC Recombination Method| Download PDF |

3. Screening of Homologous Recombinant ES cells
The ES cells are cultured on feeder cells，prepared with primary embryonic fibroblasts in either DMEM supplemented with 20% FBS and LIF (TT2 cells) or 3i medium with LIF (HK3i cells). The linealized targeting vector is electroporated into ES cells, and the cells are selected against G418. About one hundred G418 resistant colonies are isolated and trypsinized; one half of the cells in each colony is passaged into each well of 24 well plates with the feeder cells and the other half is subjected to the PCR analysis for homologous recombinant. For the PCR analysis, the cells are washed with PBS buffer, treated with protease K at 55 ℃ for 30 min, heat- treated at 95℃ for 10 min and used as templates. PCR positive colonies are passaged successively into φ22 mm, φ 35 mm, 25 cm2 and 2x25 cm2 feeders every 2-3 days and frozen at 5x106 cells per tube; the homologous recombination is confirmed by Southern blot analysis.

4. Production of Chimera Mice
Targeted ES cells are injected into ICR 8-cell stage embryos to generate chimeric mice. Usually most of the mice are 100 % ES-derived and mated with C57BL/6 to generate F1 heterozygotes.