Gelatins

The final targeting constructs were prepared for ES cell electroporation from 2 ml of culture (2X LB plus antibiotics) in 96-well format using the Qiagen Turboprep kit. Before electroporation, vectors were linearized with AsiSI and examined by gel electrophoresis. For most clones, the digested DNA migrated as a single high-molecular-mass band of the expected size (Supplementary Fig. 5). Occasionally, contaminating smaller molecular mass bands were also observed on the gel (DNA quality failures).
JM8 mouse ES cell lines derived from the C57BL/6N strain were grown either on a feeder layer of SNL6/7 fibroblasts (neomycin and/or puromycin resistant) or on gelatinized tissue culture plates^{16 (link)}. Both feeder-independent and feeder-dependent lines were maintained in Knockout DMEM (500 ml, Gibco) supplemented with 2 mM glutamine, 5 ml 100× β-mercaptoethanol (360 μl in 500 ml PBS, filter sterilized), 10–15% fetal calf serum respectively (Invitrogen) and 500 U ml⁻¹ leukaemia-inhibitory factor (ESGRO, Millipore). Trypsin solution was prepared by adding 20 ml of 2.5% trypsin solution (Gibco) and 5 ml chicken serum (Gibco) to 500 ml filter-sterilized PBS containing 0.1 g EDTA (Sigma) and 0.5 g d-glucose (Sigma).
Electroporations of ES cells were carried out in a 25-well cuvette using the ECM 630 96-well electroporator /HT-200 automatic plate handler (BTX Harvard Apparatus; set at 700 V, 400 Ω, 25 μF). Immediately before electroporation, cell suspensions of ~1 × 10⁷ cells and ~2 μg of linearized targeting vector DNA were mixed in a final volume of 120 μl PBS. Cells were seeded onto a 10-cm dish (with feeders or gelatin) and colonies were picked after 10 d of selection in 100 μg (active) per ml Geneticin (Invitrogen). To expand cells into duplicate wells for archiving and preparation of genomic DNA, confluent cultures of JM8 ES cells grown on feeder cells were washed twice with pre-warmed PBS and trypsinized for 15 min at 37 °C. Five volumes of pre-warmed media were added and the cells were gently dispersed by tituration and passed at a dilution of 1:4 into new plates containing feeder cells. Passage of cells grown on gelatinized plates was carried out in a similar manner except that the cells were trypsinized for 10 min and passed at a dilution of 1:6 into freshly gelatin-coated plates (0.1% gelatin, Sigma G1393). Culture medium was replaced daily and cells reached confluence 2 days after passage. To archive ES cell clones, trypsinized cells from confluent 96-well plates were transferred in 200 μl freezing medium (Knockout DMEM, 15% serum/ 10% DMSO) to 96-well cryovials (Matrix) and overlayed with sterile mineral oil. The cells were placed at −80 °C overnight and then transferred to liquid nitrogen.