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Targeted integration into pseudo attP sites of CHO cells using CRISPR/Cas9.
Pourtabatabaei, Sana; Ghanbari, Samaneh; Damavandi, Narges; Bayat, Elham; Raigani, Mozhgan; Zeinali, Sirous; Davami, Fatemeh.
Affiliation
  • Pourtabatabaei S; Department of Biotechnology, College of Science, University of Tehran, Tehran, Iran; Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran; Kawsar Genomics and Biotech Center, Tehran, Iran.
  • Ghanbari S; Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran.
  • Damavandi N; Department of Biotechnology, College of Science, University of Tehran, Tehran, Iran; Kawsar Genomics and Biotech Center, Tehran, Iran.
  • Bayat E; Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran.
  • Raigani M; Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran.
  • Zeinali S; Department of Biotechnology, College of Science, University of Tehran, Tehran, Iran; Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran; Kawsar Genomics and Biotech Center, Tehran, Iran.
  • Davami F; Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran. Electronic address: f_davami@pasteur.ac.ir.
J Biotechnol ; 337: 1-7, 2021 Aug 20.
Article in En | MEDLINE | ID: mdl-34157351
ABSTRACT
Chinese hamster ovary (CHO) cells are regarded as a prominent host for manufacturing therapeutic proteins. Although conventional strategies for generating recombinant proteins in CHO cells depend on the random integration of a gene of interest (GOI), these established techniques occasionally result in genetically heterogeneous cell lines, which causes diminished expression of the recombinant proteins in the long run. Production instability can be reduced by SSI and creates stable cell lines with a consistent expression of the GOI. In this experiment, we demonstrate the targeted incorporation of a reporter cassette in two PhiC31 pseudo attP sites of CHO cells exploiting the homology-directed repair (HDR) generated by the CRISPR/Cas9 platform. Genes encoding GFP and puromycin resistance marker were precisely inserted into these loci via CRISPR/Cas9. Stable cell lines were suitably produced following antibiotic selection. Junction PCR and fluorescence assay determined targeted integration and expression homogeneity of the reporter cassette, respectively. Taken together, our results indicate the possibility of these two PhiC31 pseudo attP sites as the target sites for site-specific integration of a transgene mediated by CRISPR/Cas9. Furthermore, higher knock-in efficiency and expression homogeneity was observed in the pseudo attP site associated with chromosome 6 compared to the pseudo attP site from chromosome 3.
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Full text: 1 Collection: 01-internacional Database: MEDLINE Main subject: CRISPR-Cas Systems Limits: Animals Language: En Journal: J Biotechnol Journal subject: BIOTECNOLOGIA Year: 2021 Document type: Article Affiliation country: Iran

Full text: 1 Collection: 01-internacional Database: MEDLINE Main subject: CRISPR-Cas Systems Limits: Animals Language: En Journal: J Biotechnol Journal subject: BIOTECNOLOGIA Year: 2021 Document type: Article Affiliation country: Iran