[HPRT gene knock-out from rat fetal neural stem cells].

The 3.0 kb 5′arm (long arm,LA) of rat HPRT gene knock-out vector was cut by SalⅠ from rat HPRT gene genomic bacterial artificial chromosome (BAC),and the 1.7 kb 3′arm (short arm,SA) was proliferated by PCR.Neo~r,5′arm,3′arm were sequentially cloned into pBS vector's relative restriction enzyme sites.For acquirement of tk gene,the 5′arm -Neo~r-3′arm fragment inserted into pKO vector to construct pKO-HPRT.The pKO-HPRT was linearized by NotⅠ,extracted by ethidium bromide,butanol and phenol/chloroform,and dialyzed by 0.025 μmol/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 μg/ml G418 and 0.2 μmol/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 4×10~3/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.