Application of oligonucleotides to construct a conditional targeting vector for porcine IκBα

Li,Hegang; Song,Xiaona; Yang,Feng; Bao,Hanxun; Lu,Xiaolong; Perez‑Campo,Flor   M.; Zhao,Jinshan

doi:10.3892/mmr.2017.7917

Application of oligonucleotides to construct a conditional targeting vector for porcine IκBα

Authors:
- Hegang Li
- Xiaona Song
- Feng Yang
- Hanxun Bao
- Xiaolong Lu
- Flor M. Perez‑Campo
- Jinshan Zhao
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Affiliations: College of Animal Science, Qingdao Agricultural University, Qingdao, Shandong 266109, P.R. China, Jiaozhou Bureau of Animal Husbandry and Veterinary Medicine, Qingdao, Shandong 266300, P.R. China, Stem Cell Biology Group, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Manchester M20 4BX, UK
Published online on: October 27, 2017 https://doi.org/10.3892/mmr.2017.7917
Pages: 653-659

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Abstract

Conditional gene targeting at porcine IκBα 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 IκBα. 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.

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