Target Discovery and Validation Reviews and Protocols

Keratin Transgenics and Knockouts 229
Fig. 5. (A) HM-1 ES cells plated at a low density (10×) grown on a gelatin-coated
dish. Cells require another passage to increase individual colony numbers. (B) HM1 ES
(original magnification ×10) colony after a first step gene targeting ready to be passaged
and screened for homologous recombination event by using PCR. (C) Highly differentiated
ES cells (original magnification ×25). Note the loss of discrete ES cell border, the
formation of cobblestone-like cells, and ES cells differentiating into fibroblasts that
extend outward from the colony. It is possible to attempt to rescue this colony by passaging
several times; however, it is not usually recommended.
1. Components and preparation of 50X stock solutions:
a. Dulbecco’s modified Eagle’s medium (DMEM)-Ham’s F12 (cat. no. 11320-
033, Invitrogen).
b. Neurobasal medium (cat. no. 21103-049, Invitrogen).
c. Supplement B27 (cat. no. 17504-044, Invitrogen) is 50X, so aliquot as 2 mL
and store at –20°C.
d. Bovine serum albumin (BSA) fraction V (cat. no. 15260-037, Invitrogen)-
13.3 mL/100 mL medium (1:1). Aliquot as 10 mL and store at –20°C.
e. Apo-transferrin (cat. no.11108-016, Invitrogen) 1 g/10 mL medium (1:1).
Aliquot as 0.5 mL and store at –20°C.