A splinkerette PCR-based genome walking technique for the identification of transgene integration sites in CHO cells

Identification of recombinant gene integrations sites in the Chinese hamster ovary (CHO) cell genome is increasingly important to assure monoclonality. While next-generation sequencing (NGS) is commonly used for the gene integration site analysis, it is a time-consuming and costly technique as it analyzes the entire genome. Hence, simple, easy, and inexpensive methods to analyze transgene insertion sites are necessary. To selectively capture the integration site of transgene in the CHO genome, we applied splinkerette-PCR (spPCR). SpPCR is an adaptor ligation-based method using splinkerette adaptors that have a stable hairpin loop. Restriction enzymes with high frequencies in the CHO genome were chosen using a Python script and used for the in vitro spPCR assay development. After testing on two CHO housekeeping genes with known loci, the spPCR-based genome walking technique was successfully applied to recombinant CHO cells to identify the transgene integration site. Finally, the comparison with NGS methods exhibited that the time and cost required for the analysis can be substantially reduced. Taken together, the established technique would aid the stable cell line development process by providing a rapid and cost-effective method for transgene integration site analysis. •Identification of transgene location is critical to assure monoclonality of CHO cells.•spPCR-based genome walking assay was developed and applied to recombinant CHO cells.•Python script-assisted selection of restriction enzyme can improve spPCR success rate.•spPCR saves required analysis time and cost substantially compared to current methods.•spPCR may aid cell line development by providing a simple and rapid analysis platform.