Background: The gene knockout technique is important in xenotransplantation research. Herein we have described the molecular cloning of two genes that are candidates to overcome the poor rate of homologous recombination.
Methods: Candidate cDNA fragments were amplified by polymerase chain reaction (PCR) with the corresponding primer sets deduced from a multiple alignment analysis of other mammalian genes from a cDNA library prepared from pig spleen tissue. To obtain the full-length cDNA, a 5'- and 3'-RACE PCR experiments was performed.
Results: We successfully isolated the cDNA sequences of two pig genes--BLM, a Bloom's syndrome-related gene, and UBL-1/SUMO-1--which are closely related to homologous recombination events. As a result, we verified the sequences of pig BLM and pig UBL-1/SUMO-1. The nucleic acid and amino acid coding sequence homologies of pig BLM gene with the corresponding human gene were 87.3% and 82.9%, respectively. The nucleic acid and amino acid coding sequence homologies of the pig UBL-1/SUMO-1 gene with the human gene were 96.4% and 100%, respectively.
Conclusion: Current research into homologous recombination provides the possibility for improvement of gene knockout efficiency by regulating the gene expression profiles of recombination-related genes. Transient interference with the expression of pig UBL-1/SUMO-1 and BLM is expected to improve gene targeting. The results of the present study provided important information to design siRNA knockdown vectors. They were also useful for ex ante evaluation of expression profiles of these genes in primary cultures of somatic cells, which may enhance the production of gene knockout pigs.