Isolation of Primary Fibroblasts from Mouse Embryos1.Treat 10 cm tissue culture plates with 0.1% gelatin for at least two hours before use.2.Sacrifice the pregnant female mouse (day 13 or 14 p.c.) by cervical dislocation. Dissect out the uterine horns and place into a petri dish containing PBS.3.Separate each embryo from its placenta and surrounding membranes. If desired, keep the yolk sac for genotyping.4.Wash each embryo by transferring it to a petri dish containing clean PBS.5.Using a Pipetman with the end of the tip snipped off, transfer the embryo into the barrel of a 1cc syringe fitted with an 18G 11/2" needle and, using the syringe"s plunger, force the embryo through the needle into a sterile 15 ml tube containing 1 ml of medium (DMEM, 1X GPS, and 15% FCS).6.Working in the laminar flow hood, aseptically transfer the 1 ml of embryo cells to a sterile 15 ml tube containing 10 ml of medium (DMEM, 1X GPS, and 15% FCS).7.Aspirate the gelatin from the 10 cm plates and plate out the sUSPensionofembryoniccells.8.Incubatetheplates@37oCuntilthecellshavereachedconfluence(approx.5days).(Theprimaryfibroblastswillbetheonlycellsthatattachandproliferate.)9.Atconfluence,splittheplate1:3bydoingthefollowing:a.WashtheplatetwicewithPBS.b.Add2mloftrypsinper10cmdishandincubate@37oCfor7minutes.c.Add5mlofmedium(DMEM,1XGPS,and10%FCS)totheplatetoneutralizethetrypsin.Resuspendthecellsbypipettingup-and-downseveraltimes.d.Uniformlydividethecellsamongthree10cmgelledplates,eachcontaining8mlofmedium(DMEM,1XGPS,and10%FCS).TheseplatesaredesignatedasPassageNo.1.10.Uponconfluence,harvestandfreezetheprimaryfibroblasts@afreezingdensityof3.0x107cells/ml.Reserveafractionofthecellstoseeda6cmgelleddishfromwhichDNAwillbepreparedforgenotyping(unlesstheyolksacwasusedforgenotypicanalyis).ThefrozencellsaredesignatedasPassageNo.2.FromtheLaboratoryofDr.AllanBradleyBaylorCollegeofMedicine,Houston,Texas