Journal: bioRxiv

Article Title: Multiplex genome editing eliminates the Warburg Effect without impacting growth rate in mammalian cells

doi: 10.1101/2024.08.02.606284

Figure Lengend Snippet: (A) Pyruvate sits at a branch point between fermentation through lactate dehydrogenase (Ldh) or oxidative metabolism starting with the pyruvate dehydrogenase (Pdh) complex. Pyruvate dehydrogenase kinase isoforms (Pdks) are regulated by the products and substrates of the Pdh reaction, forming a negative feedback loop that reinforces an increase in lactate secretion when glycolytic flux is high. (B) Gene expression data from major CHO cell lines shows that Pdh complex subunits are all expressed, as are all four Pdk isoforms. Ldhb shows near zero expression (0 expression in CHO-S and DG44, below 0.1 FPKM in CHO-K1 and DXB11) while Ldha expression is high. All gene expression values are given in fragments per kilobase per million reads (FPKM), data sources are provided in Supplementary Table 4. (C) Simultaneously targeting Ldha and the Pdk genes for knockout using CRISPR/Cas9 leads to no detectable Ldha expression, as verified by chemiluminescent Western blot. (D) No phosphorylation of Pdh at S293 is detected in knockout lines. (E) Total Pdh remains stable in knockout lines. (F) Enzymatic assay verifies a loss of lactate dehydrogenase activity in knockout lines (shown here as percent of wildtype Ldh activity, n=3 technical replicates per cell line). (G) Measurement of maximum lactate levels during growth in batch culture ( , clones with 4 Pdk knockouts) shows that lactate cannot be detected (n=2 shake flasks per cell line). Data shown as mean ± standard deviation (uncertainty due to variance in background in (F) accounted for via propagation of error). Gene abbreviations are as follows: Pdk1-pyruvate dehydrogenase kinase 1, Pdk2-pyruvate dehydrogenase kinase 2, Pdk3-pyruvate dehydrogenase kinase 3, Pdk4-pyruvate dehydrogenase kinase 4, Pdha1-pyruvate dehydrogenase E1 alpha 1 subunit, Pdhb-pyruvate dehydrogenase E1 beta subunit, Dlat-dihydrolipoamide S-acetyltransferase, Dld-dihydrolipoamide dehydrogenase, Ldha-lactate dehydrogenase A, Ldhb-lactate dehydrogenase B.

Article Snippet: CHO-S TM cells (Gibco Cat. # A11557-01) were transfected using either FreeStyle MAX reagent (Gibco Cat. # 16447100) or FuGENE HD reagent (Promega Cat. # E2311).

Techniques: Expressing, Knock-Out, CRISPR, Western Blot, Enzymatic Assay, Activity Assay, Clone Assay, Standard Deviation

Journal: bioRxiv

Article Title: Multiplex genome editing eliminates the Warburg Effect without impacting growth rate in mammalian cells

doi: 10.1101/2024.08.02.606284

Figure Lengend Snippet: Warburg-null cell lines (n=2 shake flasks for each clone) remain proliferative with (A) a growth rate comparable to WT while (B) drastically reducing their glucose uptake rate. This effect is observed whether in-frame mutations are counted as knockouts (left) or wildtype (right). The growth of wildtype CHO-S is shown in blue. Long-term passaging over approximately 87 days shows that Warburg-null clones (C) have higher residual glucose in culture while (D) not producing lactate and (E) maintain a comparable growth rate to WT cells (n=1 shake flask for each cell line in long-term passaging experiment).

Article Snippet: CHO-S TM cells (Gibco Cat. # A11557-01) were transfected using either FreeStyle MAX reagent (Gibco Cat. # 16447100) or FuGENE HD reagent (Promega Cat. # E2311).

Techniques: Passaging, Clone Assay

Journal: International Journal of Molecular Sciences

Article Title: MiRNAs Expression Profiling of Bovine ( Bos taurus ) Testes and Effect of bta-miR-146b on Proliferation and Apoptosis in Bovine Male Germline Stem Cells

doi: 10.3390/ijms21113846

Figure Lengend Snippet: The information of antibodies used for western blot.

Article Snippet: Bax , 1:500 , A11550 , ABclonal.

Techniques: Western Blot

Journal: Journal of Cellular and Molecular Medicine

Article Title: SV40 intron, a potent strong intron element that effectively increases transgene expression in transfected Chinese hamster ovary cells

doi: 10.1111/jcmm.13504

Figure Lengend Snippet: Effect of different introns on the production of eGFP in stable transfected CHO‐S cell. ( A ) Relative eGFP mRNA levels in stable transfected cells. At 48 hrs after transfection, stable transfected cells were selected by 800 μg/ml geneticin selection for 2 weeks until positive colonies appeared. Then, stable cell populations exhibiting stable transgene integration were cultured in CD CHO medium supplemented with 8 mM l ‐glutamine in 125‐ml Corning shake flasks with 30 ml medium with the presence of 500 μg/ml G418 for 30 days. Total RNA was isolated from 5 × 10 6 cells using the RNApure Tissue Kit, analysis of the mRNA levels was determined using real‐time quantitative PCR. ( B ) eGFP MFI was determined by cytometry for stable transfected cell pool with the different introns. Cells were collected and the eGFP fluorescence profile was measured for by FACSCalibur at 30 days after transfection. ( C ) eGFP MFI expression was normalized to IVS, and in the statistical analysis of eGFP expression, fold change values were normalized to those of the IVS, whose value was set to 1. Three independent experiments were performed in this study. Standard error of the mean (S.E.M.) is indicated (Student's t ‐test, * P < 0.05). ( D ) Percentage of high producers (% M3), fluorescence values greater than 10 4 . Three independent experiments were performed in this study. Standard error of the mean (S.E.M.) is indicated. ( E ) Coefficient of variability (CV). CV values are expected to reflect variations in transgene expression. The results are the mean values obtained for 3 independent experiments, standard deviation is indicated. ( F ) Correlation between the relative eGFP mRNA levels and relative eGFP protein expression in stable transfected CHO cell. Little correlation was noted between the eGFP mRNA levels and the eGFP protein.

Article Snippet: CHO‐S cells (Life Technologies # A11557‐01, Carlsbad, CA, USA) were grown at 37°C in a humidified incubator with 5% CO 2 , in Dulbecco's modified Eagle's medium (Gibco, Grand Island, NY, USA) supplemented with 10% foetal bovine serum (Gibco) for adherent culture.

Techniques: Transfection, Selection, Stable Transfection, Cell Culture, Isolation, Real-time Polymerase Chain Reaction, Cytometry, Fluorescence, Expressing, Standard Deviation