New strategy of constructing the β2m gene targeting vector

AIM:The purpose of this study was to establish a new strategy for constructing the mouse β₂m gene targeting vector in order to increase the homologous recombination frequency in contrast with our previous one, which was successfully constructed in the normal way.METHODS:A 4.2 kb 3' arm and a 0.8 kb 5' arm were amplified by PCR from the mouse β₂m-pSV2△HXgpt genomic clone. They included the start region and the three exons, which were separated into two parts from exons 2 (the main coding block) for the two arms——5' arm and 3' arm.RESULTS:The two fragments, in reverse orientation to the Neo gene, were cloned into pPNT respectively on either side of Neo. They were identified by PCR, restriction analysis and sequence analysis as well.CONCLUSION:The mouse β₂m gene targenting vector has been cloned successfully.