Engineering RNA viruses with unnatural amino acid to evoke adjustable immune response in mice

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   ScreenIT

   Dec 10, 2021

   SciScore for 10.1101/2021.12.04.471206: (What is this?)

   Please note, not all rigor criteria are appropriate for all manuscripts.

   Table 1: Rigor

   Ethics	IACUC: Animal experiments: All animal experiments were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee of the Peking University.
   Sex as a biological variable	Subsequently, we performed embryo transplantation into surrogate ICR females to breed the F0 generation.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	Contamination: Authentication and test for the free of mycoplasma were performed by our lab. Authentication: Virus growth curve analysis: To determine in vitro virus growth rates, triplicate wells of confluent transgenic cells, HEK293T-tRNA/PylRS/GFP39TAG, HEK293T-6stRNAGln/GFP39TAG and …

   SciScore for 10.1101/2021.12.04.471206: (What is this?)

   Please note, not all rigor criteria are appropriate for all manuscripts.

   Table 1: Rigor

   Ethics	IACUC: Animal experiments: All animal experiments were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee of the Peking University.
   Sex as a biological variable	Subsequently, we performed embryo transplantation into surrogate ICR females to breed the F0 generation.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	Contamination: Authentication and test for the free of mycoplasma were performed by our lab. Authentication: Virus growth curve analysis: To determine in vitro virus growth rates, triplicate wells of confluent transgenic cells, HEK293T-tRNA/PylRS/GFP39TAG, HEK293T-6stRNAGln/GFP39TAG and HEK293T-3CD/GFP, (6-well plate format, 106 cells/well) were infected at a MOI of 0.1.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Anti-3D antibodies (1:500, ab15277, Abcam), anti-N protein antibodies (1:500, ab7164, Abcam)	Anti-3D suggested: None anti-N protein suggested: (Abcam Cat# ab7164, RRID:AB_305740)
   , anti-GFP antibodies (1:3000, 12715-1-AP, Proteintech), anti-PylRS antibodies (1:3000, sc-365062, Santa Cruz Biotechnology, to detect Myc-tagged PylRS) were diluted in TBST containing 5% (v/v) of defatted milk.	anti-GFP suggested: None anti-PylRS suggested: (Edward A. Lemke - JGU & IMB Mainz Cat# SAR 130, RRID:AB_2893022) sc-365062 suggested: (Santa Cruz Biotechnology Cat# sc-365062, RRID:AB_10847862)
   The coated plates were then incubated with serum samples for IgM and anti-VP1 IgG, and followed by incubating HRP-conjugated anti-mouse/ferret IgG antibody or HRP-conjugated anti-mouse/ferret IgM antibody (Sino Biological Inc., Beijing, China) for 1 h at 37 °C.	anti-VP1 IgG suggested: (MyBioSource Cat# MBS432055, RRID:AB_10567698) anti-mouse/ferret IgG suggested: None anti-mouse/ferret IgM suggested: None
   The sections were incubated with anti-3D antibody (1:2000, GTX630193, GeneTex) diluted in blocking buffer at 4 °C overnight.	GTX630193 suggested: (GeneTex Cat# GTX630193, RRID:AB_2888196)
   Experimental Models: Cell Lines
   Sentences	Resources
   Then it was used as template to arrive the 12 tRNAMmPylRS copies which were then cloned into the Tol2-pylRS-GFP39TAG plasmid via Gibson assembly to obtain the Tol2-12tRNApyl-pylRS-GFP39TAG plasmid, used for NAEK-Vero stable cell line construction subsequently.	NAEK-Vero suggested: None
   Establishment of NAEK-293T stable cell line: HEK293T cells were used for lentiviral vector packaging and transduction.	NAEK-293T suggested: None HEK293T suggested: KCB Cat# KCB 200744YJ, RRID:CVCL_0063)
   The resultant stably transduced HEK293T-PylRS/GFP39TAG cells were transfected with linearized bjmu-12tRNAMmPyl -zeo plasmid DNA and cultured under the pressure of 200 µg/mL Zeocin until parental cells completely died.	HEK293T-PylRS/GFP39TAG suggested: None
   In 15 days, about 80% of the Vero cells expressed GFP fluorescence, indicating MmPylRS/tRNAMmPylRS gene was functional to read-through the amber codon introduced in GFP.	Vero suggested: CLS Cat# 605372/p622_VERO, RRID:CVCL_0059)
   2x105 cells per well from the HEK293T-tRNA/PylRS/GFP39TAG, HEK293T- 6GlnstRNA/GFP39TAG and HEK293T-3CD/GFP cell lines were seeded into six well plates in DMEM supplemented with 10% FBS for 24 h before transfection.	HEK293T-3CD/GFP suggested: None
   In brief, 5×104 Vero-E6 cells (or NAEK-Vero cells for EV71- NAEK quantification) were seeded in 96-well plates the day before infection.	Vero-E6 suggested: None
   Experimental Models: Organisms/Strains
   Sentences	Resources
   Three weeks after birth, the tail DNA of all the F0 generation mice was extracted for PCR analysis to identify the positive mice, which were further crossed with wild type C57BL/6 mice (♀) to produce the next generation.	C57BL/6 suggested: None
   For protective efficacy study, five BALB/c mice of 4-week old in each group were immunized for twice (0 and 14 days) with the same dose.	BALB/c suggested: None
   Recombinant DNA
   Sentences	Resources
   The GFP gene with an amber codon introduced at residue position 39 was expressed under a CMV promoter and cloned into the Tol2-pylRS plasmid to obtain Tol2-pylRS-GFP39TAG plasmid.	Tol2-pylRS suggested: None
   The pcDNA 3.1 vectors carrying 6 tRNAMmPylRS copies driven by human H1, human U6 and human 7sk promoters respectively were preserved in our lab.	pcDNA 3.1 suggested: RRID:Addgene_20407)
   Then it was used as template to arrive the 12 tRNAMmPylRS copies which were then cloned into the Tol2-pylRS-GFP39TAG plasmid via Gibson assembly to obtain the Tol2-12tRNApyl-pylRS-GFP39TAG plasmid, used for NAEK-Vero stable cell line construction subsequently.	Tol2-pylRS-GFP39TAG suggested: None
   Then, cells were co-transfected with 0.72 µg of pSD31 transfer plasmid, 0.64 µg of pRSV, 0.32 µg of pMD2G-VSVG and 0.32 µg of pRRE using the transfection reagent Megatran1.0 (Origene).	pSD31 suggested: None pRSV suggested: RRID:Addgene_106453) pMD2G-VSVG suggested: None pRRE suggested: None
   The resultant dual lentiviruses pSD31-PylRS and pSD31- GFP39TAG were used to integrate MmPylRS and the GFP39TAG gene into the genome of HEK293T cells.	pSD31-PylRS suggested: None pSD31- suggested: None
   The resultant stably transduced HEK293T-PylRS/GFP39TAG cells were transfected with linearized bjmu-12tRNAMmPyl -zeo plasmid DNA and cultured under the pressure of 200 µg/mL Zeocin until parental cells completely died.	bjmu-12tRNAMmPyl -zeo suggested: None
   Establishment of NAEK-Vero stable cell line: For electroporation, Vero-E6 cells were resuspended in Opti-MEM and counted, and 1×106 cells were mixed with 15 μg of linearized Tol2-12tRNApyl-pylRS-GFP39TAG plasmid in medium cup (EC002S).	Tol2-12tRNApyl-pylRS-GFP39TAG suggested: None
   Then the wild type EV71 plasmid was linearized by MluI restriction enzyme digestion.	EV71 suggested: None
   Software and Algorithms
   Sentences	Resources
   The optical bands were visualized in a Fuji Las-3000 dark box (FujiFilm), and band densities were quantified using Quantity One Analysis software.	Quantity One Analysis suggested: None
   The raw data were quality controlled by Q30 and the clean data was aligned to the reference genome (Ensembl hg38) using HISAT2 (v2.1.0)	HISAT2 suggested: (HISAT2, RRID:SCR_015530)
   Reads Count for each gene in each sample was counted by HTSeq v0.6.0, and FPKM (	HTSeq suggested: (HTSeq, RRID:SCR_005514)
   DESeq was used for differential gene expression analysis between two samples and Genes with q≤0.05 and |log2_ratio| ≥ 1 were identified as differentially expressed genes.	DESeq suggested: (DESeq, RRID:SCR_000154)
   GO and KEGG pathway enrichment analysis were performed based on differentially expressed mRNAs.	KEGG suggested: (KEGG, RRID:SCR_012773)
   All tests were two-sided and performed in GraphPad Prism 8.	GraphPad Prism suggested: (GraphPad Prism, RRID:SCR_002798)

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   Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

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   Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

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   Results from TrialIdentifier: No clinical trial numbers were referenced.

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   Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

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   Results from JetFighter: We did not find any issues relating to colormaps.

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   Results from rtransparent:

   • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
   • Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
   • No protocol registration statement was detected.

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   Results from scite Reference Check: We found no unreliable references.

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   Version published to 10.1101/2021.12.04.471206v1 on bioRxiv

   Dec 7, 2021