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Lentivirus transduction of salivary gland epithelial buds

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by Shaohe Wang

This protocol demonstrates the transduction of 20 buds with 2 different viruses (10 buds per virus). Adjust according to your needs.

  1. Materials
    • Timed pregnant mice at E13-E13.5 for isolating embryonic salivary glands
    • 70% ethanol spray bottle
    • Standard forceps (e.g. Fine Science Tools, 11000-12)
    • Fine sharp scissors (Fine Science Tools, 14060-10)
    • DMEM/F-12 (e.g., Thermo Fisher, 11039047)
    • 100x PenStrep (10,000 units/mL penicillin, 10,000 µg/mL streptomycin; e.g., Thermo Fisher, 15140163)
    • DMEM/F-12 + 1x PenStrep (referred to as “medium” throughout this protocol): add 5 mL 100x PenStrep to 500 mL DMEM/F-12
    • Dissecting microscope (e.g., Leica M80; any common dissecting microscopes should work)
    • Dumont #5 forceps (Fine Science Tools, 11251-20)
    • Dumont #5 fine forceps (Fine Science Tools, 11254-20)
    • Scalpel (Fine Science Tools, 10011-00 and 10003-12)
    • 10-cm dish (e.g., Corning 430167)
    • 35-mm dish (e.g., Corning, 430165)
    • Pyrex spot plate (Fisher Scientific 13-748B)
    • 22x22 mm coverslip (e.g., MilliporeSigma, C9802)
    • Dispase (Thermo Fisher, 17105041); dilute in DMEM/F-12 medium at 5-10 U/mL, aliquot and store at -20°C
    • 5% BSA (MilliporeSigma, A8577; diluted in DMEM/F-12)
    • 200 µL low-retention pipette tips (Rainin, 30389187)
    • 20 µL low-retention tips (Rainin, 30389190)
    • 20 µg/mL FGF7 (R&D Systems, 5028-KG-025); divide into 25 µL aliquots and store at -20°C
    • 100x ITS supplement (Thermo Fisher, 41400045)
    • Growth factor-reduced Matrigel (Corning, 356231; stock is usually 9-10 mg/mL)
    • Ultra-low attachment 96-well V-bottom plate (S-bio, MS-9096VZ)
    • Concentrated virus stocks at -80°C. See Lentivirus packaging.
    • Polybrene (4 mg/mL in water; Millipore- Sigma, H9268)
  2. Thaw at least 105 µL Matrigel on ice or in a fridge before dissection.
    • Each bud will need 5 µL Matrigel stock for culture. Adjust amount based on your needs.
    • Matrigel stock has to be thawed at low temperature to prevent premature solidification, and it can take several hours to completely thaw.
  3. Please follow the protocols below to obtain single epithelial buds of embryonic salivary glands.
  4. Thaw the two viruses stored at -80°C, when buds are ready and stored in 5% BSA in medium.
    • Each bud will need 10 µL virus stock, so make sure there is at least 105 µL of each virus.
    • The titer of the virus stock is usually ~1.5 x 108 IFU/mL. See Lentivirus packaging.

  5. Dilute 1 µL 4 mg/mL polybrene 1:25 in medium to obtain 160 µg/mL polybrene.

  6. Make a master mix of each virus. Each bud will be treated in a final volume of 20 µL with 8 µg/mL polybrene in a well of the ultra-low attachment 96-well V-bottom plate. 5 µL carryover volume is assumed for each bud during transfer.

    Stock µL for one well 10.5x (make 0.5 extra)
    virus stock 10 105
    160 µg/mL polybrene 1 10.5
    medium 4 42

  7. Prepare an ultra-low attachment 96-well V-bottom plate by filling in different amounts of medium using a multichannel pipette:

    Wells Usage µL of medium to fill
    A1-H1 & A12-H12 edge wells to fill in medium to prevent evaporation 150
    A2-A11 virus 1 treatment 0
    B2-C11 virus 1 washes 150
    D2-D11 culture of virus 1 treated buds 47
    E2-E11 culture of virus 2 treated buds 47
    F2-G11 virus 2 washes 150
    H2-H11 virus 2 treatment 0
  8. In the Pyrex 9-well glass spot plate (Fisher Scientific, 13-748B) used for dissection, wash single epithelial buds twice in medium without BSA.
    • Use 20 µL low-retention tips (cut for larger opening; Rainin, 30389190) to transfer buds.
  9. Transfer one bud into each well for virus treatment with 5 µL carryover volume.
    • These are wells A2-A11 and H2-H11 of the ultra-low attachment 96-well V-bottom plate).
    • Use 20 µL low-retention tips (cut for larger opening; Rainin, 30389190) to transfer buds.

  10. For each bud, treat with 15 µL virus master mix containing polybrene.

  11. For transduction, incubate the plate in a humidified 37°C incubator for 1-2 hours. The incubator does not need to have CO2.

  12. During the incubation, prepare 2x culture mix.

    Stock µL for one well 21x (make one extra)
    Medium 43 903
    FGF7 (20 µg/mL, 100x) 1 21
    ITS (100x) 1 21
    Matrigel (~10 mg/mL) 5 105
  13. After the incubation, wash off the virus by transferring the bud into the wash wells containing 150 µL medium.
    • During the incubation, it is normal that viral particles precipitate and form a thin film that appears brownish under the dissecting microscope. Washing off the film requires thorough trituration.
    • Use 20 µL low-retention tips (cut for larger opening; Rainin, 30389190) to transfer buds.
    • Triturate thoroughly in each well for the wash.
  14. After the washes, transfer the bud into the 47 µL medium in the culture wells with 3 µL carryover volume.
    • Use 20 µL low-retention tips (cut for larger opening; Rainin, 30389190) to transfer buds.

  15. To each culture well, add 50 µL 2x culture mix prepared above.

  16. Incubate the plate at 37°C with 5% CO2.
    • Fluorescence should be readily detectable after 24 hours of culture.
    • For CRISPR/Cas9-mediated knockout, culture for 2-3 days before analysis of protein expressing and/or phenotype.