Journal: Scientific Reports

Article Title: Glutamine synthetase (GS) knockout (KO) using CRISPR/Cpf1 diversely enhances selection efficiency of CHO cells expressing therapeutic antibodies

doi: 10.1038/s41598-023-37288-6

Figure Lengend Snippet: The endogenous GS genes in CHO-K1 genome database and possible pgRNA for knockout the GS genes. ( A ) Schematic representation of GS genes in CHO-K1 genome and sgRNA positions. The sgRNA positions are shown as vertical lines. The solid arrows indicate primers to detect the deletion PCR product and dashed arrows indicate primers to detect the non-deletion PCR product. Color of arrows represent each pair of PCR reaction. ( B ) Expression of various GS genes in CHO-K1 and S. Relative mRNA expression of two GS genes in CHO-K1 and S, normalized to GAPDH, are shown. The data represent mean ± SD from a quadruplicate representative example. Statistical analysis was performed with the GraphPad Prism 8.0.1 software using Student’s t -test and followed by Welch’s test. Symbol “ a , b , c ” indicate a statistically significant difference p < 0.001, 0.01 and 0.05, respectively and ns indicates no significant difference. ( C ) Evaluation of pgRNA efficiency for GS gene deletions. Each vector carrying pgRNA was transfected into CHO-K1. After transfection, the transfected cells were tested for gene deletion by PCR. The pool cells showing the deletion amplicon indicated that the designed sgRNA pairs can be used to create GS-KO CHO cells. The illustrated gel is the representative example of one biological replicate. ( D ) Assessment of pgRNA efficiency for the deletion of GS1 gene in sGS5KO-K and sGS5KO-S cells by deletion PCR. The original gels are presented in Supplementary Fig. ).

Article Snippet: Graph and Statistic analysis was performed using GraphPad Prism 8.0.1 software ( https://www.graphpad.com/updates/prism-801-release-notes?fbclid=IwAR0SMvrkrXR6EAnVoV3klghG7Wa614o519u0CEcfYmzDDEeL5OVF6KOUdpo ).

Techniques: Knock-Out, Expressing, Software, Plasmid Preparation, Transfection, Amplification

Journal: Scientific Reports

Article Title: Glutamine synthetase (GS) knockout (KO) using CRISPR/Cpf1 diversely enhances selection efficiency of CHO cells expressing therapeutic antibodies

doi: 10.1038/s41598-023-37288-6

Figure Lengend Snippet: Growth behavior of GS-KO CHO cell lines. ( A ) The cells were cultured in medium supplemented with L-Gln. ( B ) The cells were cultured in medium without L-Gln. All values represent mean ± S.D. of triplicates. Statistical analysis was performed with the GraphPad Prism 8.0.1 software using two-way ANOVA and followed by Dunnett’s multiple comparison test. Symbol “ a , b , c ” indicate a statistically significant difference versus wild type (WT) p < 0.001, 0.01 and 0.05, respectively.

Article Snippet: Graph and Statistic analysis was performed using GraphPad Prism 8.0.1 software ( https://www.graphpad.com/updates/prism-801-release-notes?fbclid=IwAR0SMvrkrXR6EAnVoV3klghG7Wa614o519u0CEcfYmzDDEeL5OVF6KOUdpo ).

Techniques: Cell Culture, Software