Objective To construct an eukaryotic expression vector of rat aquaporins (AQP4)-M1 and to establish its stable transfectants in CHO cells. Methods The fragment of rAQP4-M1 was amplified from rat brain by RT-PCR

and then the PCR products were cloned into pcDNA3.1(+) after

Nde

Ⅰ and

Xho

Ⅰ digestion. The eukaryotic expression vector pcDNA3.1(+)/rAQP4-M1 was sequenced and transfected into CHO cells by Lipofectamine 2000. After screening by G418

stably transfected CHO cell line was established. The expression of rAQP4-M1 was identified by immunocytochemistry and immu

noblotting. Blue-native polyacrylamide gel electrophoresis (BN-PAGE) was used to detect the formation of orthogonal arrays of particles (OAPs). Results The eukaryotic expression plasmid of pcDNA3.1(+)/rAQP4-M1 was successfully constructed and its stable transfectants in CHO cells was established. Immunofluorescence image revealed that rAQP4-M1 was expressed successfully on the membrane of CHO cells. Immunoblotting results following SDS-PAGE indicated the positive band was at 34×10

3

which manifesting that rAQP4-M1 was successfully and stab1y expressed in CHO cells. Results of BN-PAGE indicated that it was rAQP4-M23 not rAQP4-M1 that played a role in the formation of OAPs. Conclusion The recombinant eukaryotic expression vector of pcDNA3.1(+)/rAQP4-M1 is constructed successfully and stab1y expressed in CHO cells

which provides a foundation for further studies on the function of AQP4 isoforms of M1 and M23.