Using a reverse genetics system to generate recombinant SARS-CoV-2 expressing robust levels of reporter genes

  1. Chengjin Ye
  2. Luis Martinez-Sobrido

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Abstract

Reporter-expressing recombinant virus represents an excellent option and a powerful tool to investigate, among others, viral infection, pathogenicity, and transmission, as well as to identify therapeutic compounds that inhibit viral infection and prophylactic vaccines. To combat the still ongoing coronavirus disease 2019 (COVID-19) pandemic, we have established a robust bacterial artificial chromosome (BAC)-based reverse genetics (RG) system to rapidly generate recombinant severe acute respiratory syndrome coronavirus 2 (rSARS-CoV-2) to study the contribution of viral proteins in viral pathogenesis. In addition, we have also engineered reporter-expressing recombinant viruses in which we place the reporter genes upstream of the viral nucleocapsid (N) gene to promote high levels of reporter gene expression that facilitates the study of SARS-CoV-2 in vitro and in vivo . Although successful, the genetic manipulation of the BAC containing the entire SARS-CoV-2 genome of ∼30,000 nucleotides, is challenging. Herein, we depict the technical details to engineer rSARS-CoV-2 expressing reporter genes using the BAC-based RG approach. We describe i) assembly of the full-length (FL) SARS-CoV-2 genome sequences into the empty pBeloBAC, ii) verification of the pBeloBAC-FL, iii) cloning of a Venus reporter gene into the pBeloBAC-FL, and iv) recovery of the Venus-expressing rSARS-CoV-2. By following this protocol, researchers with basic molecular biology and gene engineering techniques knowledge will be able to generate wild-type and reporter-expressing rSARS-CoV-2.

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  1. ScreenIT

    SciScore for 10.1101/2022.05.21.492922: (What is this?)

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

    Table 1: Rigor

    Ethicsnot detected.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    (iv) Wash the cells 3 times with PBS. (v) Dilute the TRITC-labeled donkey anti-mouse IgG secondary antibody in blocking solution to a final concentration of 1 µg/mL, and incubate with the cells (1 mL/well) at 37°C for 1 h.
    anti-mouse IgG
    suggested: None
    (iv) Wash the cells 3 times with PBS. (v) Dilute the biotinylated horse anti-mouse secondary antibody in blocking solution, and incubate with the cells (1 mL/well) at 37°C for 1 h.
    anti-mouse
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Rescue of rSARS-CoV-2 using the pBeloBAC-FL: Transfection of the pBeloBAC-FL into Vero E6 cells: 33.
    Vero E6
    suggested: RRID:CVCL_XD71)
    Recombinant DNA
    SentencesResources
    CRITICAL The pBeloBAC plasmids that are used for cloning must be purified.
    pBeloBAC
    suggested: None
    The PCR-positive clone is further confirmed by sanger sequencing using the CMV/F and bGH/R primers (Table 2) and designated as pBeloBAC-F1.
    pBeloBAC-F1
    suggested: None
    Set up a restriction digestion of PacI and MluI to linearize the purified pBAC-F1, similar to step 13.
    pBAC-F1
    suggested: None
    Next, assemble F4 (using MluI and BstBI), F2 (using KasI and PacI), and F5 (using BstBI and BamHI) into pBeloBAC-F13 one by one as previously described for F3 into pBeloBAC-F1.
    pBeloBAC-F13
    suggested: None
    The resultant plasmids are named, respectively, pBeloBAC-F134, pBeloBAC-F1342, and finally pBeloBAC-FL, which contains F1, F2, F3, F4 and F5.
    pBeloBAC-F134
    suggested: None
    pBeloBAC-F1342
    suggested: None
    CAUTION Transfection to generate rSARS-CoV-2 using the pBeloBAC-FL must be performed in a biosafety level (BSL) 3 laboratory following institutional biosafety guidelines.
    rSARS-CoV-2
    suggested: None
    Cloning of the Venus reporter gene into the pBeloBAC-FL: Cloning of Venus into the pUC57-F1: 47.
    pBeloBAC-FL
    suggested: None
    Set up a PCR reaction in a 0.2 mL tube according to the list below to amplify pUC57F1.
    pUC57F1
    suggested: None
    The positive plasmid is designated as pUC57-F1/Venus-2A.
    pUC57-F1/Venus-2A
    suggested: None
    The positive clone is named as pBeloBAC-FL/Venus-2A.
    pBeloBAC-FL/Venus-2A
    suggested: None

    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).

    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.

    Results from TrialIdentifier: No clinical trial numbers were referenced.

    Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

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

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

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

    About SciScore

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  2. Version published to 10.1101/2022.05.21.492922v1 on bioRxiv