ObjectiveTo investigate the function of a novel mouse imprinting gene which was cloned by DD -PCR and RACE, a knockout strategy was applied to target this gene, so in this study the targeting vector was constructed and the recombinant cell clones were primarily screened by PCR. Methods Two large fragments flanking the coding region of this gene were cloned into the pKO Scrambler NTKV -1906 vector after the genomic sequence of this gene was analyzed by computer, and then the linearized replacement vector was transfected into the mouse ES cells and drug -resistant cell clones were picked out for screening by a PCR -based strategy. Results The targeting vector and a control vector were constructed successfully and three recombinants were obtained by PCR screening for further use. Conclusion This targeting vector and the recombinants provided a support for identification of the function of the mouse novel imprinting gene . At the same time, a PCR -based screening was established and successfully applied to obtain three positive clones for further use, which would reduce the work of screening in the knockout process.