Schematic diagram depicting gene trap screening strategy. Two gene trap vectors were used. The PT1 vector contains a promoterlesslacZ gene immediately downstream of a splice acceptor (SA) site and the neo^R gene driven by the PGK-1promoter. Although not all neo^R colonies represented trapped genes, all genes could be trapped regardless of their expression in undifferentiated ES cells using the PT1 vector. The GT1.8geo vector contains a promoterless lacZ-neo^R(β-geo) fusion gene immediately downstream of an SA site. Although all neo^R colonies represented trapped genes, only genes expressed in undifferentiated ES cells could be trapped. ES cells were electroporated with either vector and G418^R clones were picked into 96-well plates and grown to confluency. The clones were passaged 1:3 into two 96-well plates and one set of 24-well plates. The cells from one 96-well plate were frozen, and the cells from the second 96-well plate were assayed forlacZ expression. The colonies in the 24-well plates were treated with dispase and then transferred to suspension 24-well plates and grown in suspension for 3 days; EBs were then transferred to 48-well tissue culture (TC) plates. Cultures were fed every other day and analyzed for lacZ expression 8, 12, and 16 days after dispase treatment. Clones exhibiting expression patterns were thawed and grown for RNA isolation, RACE polymerase chain reaction (PCR) and sequencing, and/or OP9 coculture and subsequent lacZexpression, and/or diploid aggregation for in vivo lacZexpression analysis and germline transmission.