A system for the measurement of gene targeting efficiency in human cell lines using an antibiotic resistance—GFP fusion gene

Abstract

Gene targeting in a broad range of human somatic cell lines has been hampered by inefficient homologous recombination. To improve this technology and facilitate its widespread application, it is critical to first have a robust and efficient research system for measuring gene targeting efficiency. Here, using a fusion gene consisting of hygromycin B phosphotransferase and 3′-truncated enhanced GFP (HygR-5′ EGFP) as a reporter gene, we created a molecular system monitoring the ratio of homologous to random integration (H/R ratio) of targeting vectors into the genome. Cell clones transduced with a reporter vector containing HygR-5′ EGFP were efficiently established from two human somatic cell lines. Established HygR-5′ EGFP reporter clones retained their capacity to monitor gene targeting efficiency for a longer duration than a conventional reporter system using an unfused 5′ EGFP gene. With the HygR-5′ EGFP reporter system, we reproduced previous findings of gene targeting frequency being up-regulated by the use of an adeno-associated viral (AAV) backbone, a promoter-trap system, or a longer homology arm in a targeting vector, suggesting that this system accurately monitors H/R ratio. Thus, our HygR-5′ EGFP reporter system will assist in the development of an efficient AAV-based gene targeting technology.

Keywords

• gene editing
• genome modification
• homologous recombination
• adeno-associated virus