Abstract: Conditional gene targeting at porcine I?B? may be a solution to delayed xenograft rejection, the main barrier to xenotransplantation. An oligonucleotide?based method was applied to construct the vector for conditional targeting of porcine I?B?. This method was free from PCR amplification during the assembling of the different vector elements, avoiding introduction of unwanted mutations. With the help of short double?stranded DNA fragments produced by annealing oligonucleotides, nondirectional cloning has also been avoided. By making the best of directional cloning, a highly complex targeting vector was built within 3 weeks. The present study also explained why the two recombination?based methods (recombineering and gateway recombination), although having demonstrated to be highly efficient in constructing ordinary targeting vectors, were not appropriate in this context. The description in the present study of an additional method to efficiently construct targeting vectors is suggested to introduce more flexibility in the field therefore helping to meet the different needs of the researchers.