Objective  To construct the eukaryotic expression vector of apolipoprotein E (APOE) ε2 and ε4 and establish APOE subtype gene-modified neural stem cells (NSCs) based on the clone of human APOE ε3 gene.   Methods  APOE ε3 cDNA was cloned in our previous work. Site-directed mutagenesis was used to obtain cDNA encoding APOE ε2 and ε4 isforms. The APOE ε2 and ε4 isforms were inserted in the eukaryotic expression vector pEGFP-N1. Primary NSCs were isolated

cultivated and purified from rat embryo pallium of APOE knock-out mice. Recombinant plasmid was transfected into NSCs with Lipofectamine 2000 and selectively cultured with G418. After NSCs expressed the EGFP gene and APOE

they were amplified. Expression of target gene in the amplified NSCs was analyzed by RT-PCR

Western blotting and laser confocal microscopy

respectively.   Results  The eukaryotic expression vectors of APOEε2 and APOEε4 were constructed. The plasmids were transfected into NSCs by Lipofectamine 2000(40.0±2.3)%. The NSCs could express EGFP after transfection. Some clones expressing EGFP were formed after selective culture with G418 at 10 day. The differented rate of neuronis (32.15±2.61)%. RT-PCR

Western blotting and laser confocal microscopy demonstrated that the human APOE gene isoforms (ε2

ε4) were expressed in NSCs.   Conclusion  Eukaryotic expression vector of APOE (ε2

ε4) can be transfected into NSCs and multipotent NSCs expressing target gene can be obtained and are able to renew

thus laying a foundation for further studies on the function of APOE subtype.