中国生物工程杂志

The expression cell lines constructed by random integrating target gene into mammal cell’s genome may not express the target gene stably over passages because the target gene might be inserted into unstable region of chromatin, which is known as position effect. To solve this problem, site specific integration of target gene (Human serum albumin gene) into stable hot spot of CHO chromatin by using homologous dependent recombination (HDR) method mediated by CRISPR/Cas9 can be effective, because the position effect issue can be overcome. Here, two site specific integration hits of human serum albumin gene were obtained verified by conducting 5' junction PCR, 3' junction PCR and out-out PCR. The Western blot results revealed target protein could be detected in the supernatants of culture; the average amount of HSA protein expressed per cell per day was around 0.5pg cell/d over different cell passages (passage 3, 12, 23, 35, 50) at adherent cell mode for both two hits. One hit was adapted to suspension culture. The expression level of this hit at batch mode in different cell passages (passage 1, 25, 50) were stably around 13-14mg/L.It was feasible to insert heterogenous gene into the stable hot spot of CHO cell line and corresponding gene expression level was stable over passages.