Derivation of ES cell lines from Mouse Blastocysts Adapted from Gene Targeting in the Mouse (Joyner) IDDRC Mouse Gene Manipulation Facility Day 1: Mate 10 – 20 male mice (age 8 weeks to 5 months) each with 2 estrus-staged females (5 – 10 weeks). Day 2 (0.5 pc): Check for plugs and separate plugged females into own cage. Day 5 (3.5 pc): Flush blastocysts from uterine horns in regular ES media, or DMEM with 10% FCS and pen/strep. Place each blastocyst in one well of a 4 well plate preplated with arrested MEFs in “HighTec” ES media (25% FCS, 2000 u/ml LIF). Day 9 – 12: Pick ICM’s into HighTec with MEK1 inhibitor (0.05 mM PD98059) each in one well of 6 well plate with feeders. In hood, with microscope, remove media. Wash 2X with 150 ul 1X PBS, leaving 2nd wash in well. Transfer by mouth pipet into 25 ul 1% chicken serum/0.25% Trypsin in 96 well. Incubate 37C, 5 minutes Add 50 ul media Pipet up and down 8X and transfer everything to 6 well well. Day 13 –14: Look at wells everyday to follow development of clones. Pick “ES” like colonies into 24 well wells with feeders in HighTec (?MEK inhibitor). Do this picking as pick for transfection. Wash plate with 1XPBS. Add Trypsin to plate and aspirate to just enough to keep plate moist (100 ul?) Incubate 2 minutes 20 seconds at 37 C. Take up small amount of trypsin in P200 tip. Drop a bit onto colony, and pipet up colony and transfer to a 96 well with 20 ul. Let sit in hood for 5 (?) minutes. Pipet up and down about 5X and transfer to 24 well well. Day 15 or 16: When colonies grow, trypsinize the usual way in same well, now with no MEK inhibitor, but still HighTec. Day 17 or 18: When confluent, split each 24 well well to 6 well well with feeders in 20% FCS, 1500 U/ml LIF ES media. Day 19 or 20: When confluent, split each 6 well well to 10 cm plate with feeders in 15% FCS, 1000 U/ml LIF (normal ES media). Day 21 or 22: Freeze cells in multiple vials. At a later point, thaw one vial from each line, expand cells, refreeze, karyotype and prepare DNA to PCR for the Y chromosome. Male cells are preferred over female cells. Each putative line will need to be injected into mouse embryos to assess germline potential.