Journal: ACS Synthetic Biology
Article Title: Engineering an Autonucleolytic Mammalian Suspension Host Cell Line to Reduce DNA Impurity Levels in Serum-Free Lentiviral Process Streams
doi: 10.1021/acssynbio.3c00682
Figure Lengend Snippet: During lentivirus production, NuPro-2S cells give rise to medium-resident nuclease activity that reduces DNA impurity levels in situ . (A) Before and after the Figure C Benzonase addition, 10 mL samples of HEK293TS cells were centrifuged at 500 rcf for 5 min, and the supernatant was removed and retained as “Clarified Media”. Uncentrifuged samples were also taken and retained as “Whole Culture”. Equivalent NuPro2S samples were also taken. All samples were then incubated at 37 °C for a 1 h hold step. This diagram illustrates the anticipated presence of nuclease activity (red symbols) arising from NuPro-2S and the presence of lentivirus (green symbols) produced from both cell lines and confirmed in Figure . (B) “Clarified Media” samples (8 μL) were incubated with 2 μL of a 500 ng/μL 1 kb DNA ladder solution for 1 h prior to agarose gel electrophoresis. Samples were either pre- or posthold, as indicated, and taken from cultures of the indicated cell lines. Cultures had either had no additions or addition of tetracycline or Benzonase as detailed in Figure . (C) 40 μL samples, either pre- or posthold, from the indicated cultures were analyzed directly by agarose gel electrophoresis. (D) 100 μL serially diluted samples posthold from the indicated cultures were analyzed using the PicoGreen reagent, and the DNA was content plotted. Error bars represent the standard deviation of means arising from samples from n = 2 lentiviral production runs, each of which was used for n = 3 Pico Green-based determinations. Significance was determined by the unpaired Student’s t test. DNA concentration differences between unsupplemented HEK293TS and Benzonase-supplemented HEK293TS ( p = 0.0921), and NuPro-2S ( p < 0.0001) were significant (asterisks), while differences between Benzonase-supplemented HEK293TS and NuPro-2S were not (ns). All gel images are representative of duplicate gels used for analysis of duplicate samples.
Article Snippet: The following third-generation lentivirus plasmids were used: pLJM1-eGFP (Addgene plasmid no. 19319) encoding the eGFP reporter payload genome, pMDLg/pRRE (Addgene plasmid no. 12251) encoding gagpol proteins, pRSV-Rev (Addgene plasmid no. 12253) encoding rev, and pMD2.G (Addgene plasmid no. 12259) encoding the VSV-G envelope protein.
Techniques: Activity Assay, In Situ, Incubation, Produced, Agarose Gel Electrophoresis, Standard Deviation, Concentration Assay
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