The 3.0 kb 5' arm (long arm, LA) of rat HPRT gene knock-out vector was cut by Sal I from rat HPRT gene genomic bacterial artificial chromosome (BAC) , and the 1.7 kb 3' arm (short arm, SA) was proliferated by PCR. Neo', 5' arm, 3' arm were sequentially cloned into pBS vector's relative restriction enzyme sites. For acquirement of tk gene, the 5' arm -Neo' -3' arm fragment inserted into pKO vector to construct pKO-HPRT. The pKO-HPRT was linearized by Not I, extracted by ethidium bromide, butanol and phenol/chloroform, and dialyzed by 0.025 microlmol/L Millipore. At the same time, rat neural stem cells cultured from E14.5-16.5 rat fetal brain. Passage 2 rFNSCs was tranfected by linearized pKO-HPRT with Fugene-6t transfection reagents. After 80 microg/ml G418 and 0.2 micromol/L ganciclovir selection, the survived cells was cultured in suspension to form neural spheres. The spheres can be picked up under the microscopy, and proliferated in 96- ,48- and 24-well plates sequentially. When the cell number reached 4 x 10(3)/well, half cells was lysed by lysis buffer to extract DNA, the other half was kept on growing to freeze and extract RNA. The knock-out cell colonies first detected by PCR, then confirmed by Southern blot and RT-PCR. All the results show that we have knocked out HPRT gene in three rat fetal neural stem cell colonies from 32 colonies.