The single transmembrane segment of gp210 is sufficient for sorting to the pore membrane domain of the nuclear envelope

The expression of chimeric proteins in mouse Baib/c 3T3 cells was accomplished by direct microinjection of plasmid DNA into nuclei using a procedure described by Capecchi (5) . Subconfiuent cultures of Baibtc 3T3 cells wore grown on 12-ram diam. glass coverslips in DME medium (Gibco Laboratories, Grand Island, NY) supplemented with 10% FBS (Gibco Laboratories), 20 mM Hepes, pH 7.4, and 50 ~g/mi Gentamycin (Gibco Laboratories). Nuclei were injected with plasmid DNA purified using a plasmid purification kit (Qingen Inc., Chatsworth, CA) at 1 mg/ml in PBS containing 1 mM MgC12. The microinjection equipment consisted of an Eppendorf injector 5,242 and a Zeiss micromanipulator MR mounted on a Zeiss Axiovert 10 microscope (Zeiss, Oberkochen, Germany). After injection, the cells were returned to a 5% CO2 incubator at 37~ for 4-5 h.