TITLE: Stable Transfection of Plasmid DNA into Adherent Rodent Cell Lines Using Calcium Phosphate
AUTHORS: Vipasha Dwivedi, Jen Davis, Ella Wilson, Markelle Scott, Madison Zelan, Nicholas DeFeo, Victoria Huffman, Rees Cooke, Kaitlyn O'Daniel, David J Riese II

[DESCRIPTION]
A traditional strategy for stably transfecting DNA into rodent fibroblast cell lines features calcium phosphate precipitates.  Here we describe our laboratory protocol for this strategy.  We have assumed that the transfected DNA contains an expression vector for an antibiotic resistance gene, such as the neomycin resistance gene, the hygromycin resistance gene, the puromycin resistance gene, and the like.

[STEPS]
SECTION: Introduction
1. A traditional strategy for stably transfecting DNA into rodent fibroblast cell lines features calcium phosphate precipitates.  Here we describe our laboratory protocol for this strategy.  We have assumed that the transfected DNA contains an expression vector for an antibiotic resistance gene, such as the neomycin resistance gene, the hygromycin resistance gene, the puromycin resistance gene, and the like.
SECTION: Methods
2. Cell Lines and Cell Culture
SECTION: Methods
2.1. NIH/3T3 [1] and C127 [2] adherent rodent cell lines are a generous gift from Daniel DiMaio.  PA317 [3], Psi-2 [4], and Psi-Cre [5] recombinant retrovirus packaging cell lines are derived from NIH/3T3 cells and are a generous gift from Daniel DiMaio [6].  
SECTION: Methods
2.2. The cell line to be transfected is typically maintained in T-75 (nominally 75 cm2) cell culture flasks according to published or vendor recommendations [1-5, 7].  The cell line is transfected no less than two passages and no greater than five passages after being established from a frozen vial of archived cells.
SECTION: Methods
2.3. Cells to be transfected should be seeded at three densities to account for experiment-to-experiment variations in cell growth rate and plating efficiency.  Thus, for each transfection, 1x105, 2x105, and 3x105 cells are seeded into a 60 mm (diameter) cell culture dish.  In other words, three dishes of cells, each at a different density, are prepared for each transfection.  Prepare enough plates of cells to allow for mock and control transfections.  Cells are incubated under standard conditions for 24-48 hours to allow cell recovery.  Cells should be at 50% confluence at the time of transfection.
SECTION: Methods
3. Preparation for DNA Transfection
SECTION: Methods
3.1. Two hours prior to transfection, the set of plates that are closest to 50% confluence are selected, while the other sets of plates are discarded.  The medium should be changed on the selected set of plates using 5 mL of standard (pre-warmed to 37oC) culture medium and the cells should be returned to the 37oC incubator.
SECTION: Methods
3.2. During this two-hour incubation, a sterile 5 mL snap-cap tube (Falcon 352063; VWR 60819-728 [8]) should be labeled for each transfection, including mock and control transfections.  
SECTION: Methods
3.3. In a biosafety cabinet, 200 uL of 2x HEPES-buffered saline (2x HEBS – see recipe below) should be added to each 5 mL snap-cap tube.
SECTION: Methods
3.4. In a biosafety cabinet, add 10 ug of uncut plasmid DNA to the appropriate 5 mL snap-cap tube(s).
SECTION: Methods
3.5. Using sterile technique, make a fresh solution of 250 mM CaCl2 by diluting sterile 2 M CaCl2 with deionized water.  Each transfection requires 200 uL of 250 mM CaCl2.  The 250 mM CaCl2 solution should be formulated in a sterile 1.5 mL microcentrifuge tube or a sterile 50 mL centrifuge tube to facilitate sterile pipetting.
SECTION: Methods
3.6. Prepare the calcium phosphate precipitates of DNA in a biosafety cabinet.  Approximately 90 minutes after changing the medium on the cells to be transfected, use a micropipetter to add 200 uL of 250 mM CaCl2 to each HEBS/DNA solution while simultaneously blowing air bubbles into the HEBS/DNA solution using a pipetter and a 1 mL sterile serological pipette.  This facilitates formation of calcium phosphate precipitates of DNA.
SECTION: Methods
3.7. Vortex to mix the precipitates and incubate for 20 minutes at room temperature in the biosafety cabinet.
SECTION: Methods
4. DNA Transfection and Shock
SECTION: Methods
4.1. Drip each precipitate onto a 60 mm dish of cells with swirling.  Confirm the presence of precipitates by microscopic inspection.  Incubate 4-6 hours at 37oC. 
SECTION: Methods
4.2. Shock cells as follows:
-       Prepare the glycerol/HEBS shock solution using a 1:1 mixture of 30% glycerol and 2x HEBS
-       Prepare complete (pre-warmed to 37oC) culture medium containing 5 mM NaButyrate.
-       Aspirate medium from each plate.
-       Add 0.7 mL glycerol/HEBS solution and incubate 45 seconds.
-       Aspirate glycerol/HEBS solution and wash twice with PBS.
-       Add 4 mL complete culture medium supplemented with 5 mM NaButyrate.
-       Incubate 12-16 hours at 37oC.
SECTION: Methods
5. Passage Cells and Select for Transfected Cells
SECTION: Methods
5.1. Use standard cell culture techniques to subculture each 60 mm dish of transfected cells into three 100 mm (diameter) cell culture dishes.
SECTION: Methods
5.2. Incubate 16-24 hours at 37oC.
SECTION: Methods
5.3. Select for transfected cells using the appropriate antibiotic. 
SECTION: Methods
5.4. Depending on the antibiotic, colonies of stably transfected (antibiotic-resistant) cells typically become apparent following 7-12 days of selection.
SECTION: Buffer Recipes
6. 2x HEBS (250 mL recipe)
SECTION: Buffer Recipes
6.1. Add the following to a 500 mL Erlenmeyer flask on the benchtop
     118 mg Na2HP04-7H20 (62.5 mg anhydrous)
     0.5 g Dextrose (D-glucose)
     4 g NaCl
     185 mg KCl
     2.5 g HEPES
Add 230 mL deionized water; stir until the solids are dissolved
Adjust pH to 7.05 w/10 N NaOH
Repeat pH next day (use concentrated HCl if pH is too high)
Adjust volume to 250 mL using deionized water
Sterile filter using a 0.2 um filter and aliquot 10 mL into twenty-five 15 mL screw top tubes
Store at -20o C until use
SECTION: Buffer Recipes
7. 1 M NaButyrate (NaC4H7O2 – 250 mL recipe)
SECTION: Buffer Recipes
7.1. Add the following to a 500 mL Erlenmeyer flask in a fume hood
     200 mL deionized water
     27.5 g NaButyrate
Stir until the solid is dissolved 
Adjust volume to 250 mL using deionized water
Sterile filter using a 0.2 um filter into a sterile 250 mL media bottle 
Store at 4o C until use
SECTION: Buffer Recipes
8. 30% Glycerol (250 mL recipe)
SECTION: Buffer Recipes
8.1. Add the following to a 500 mL Erlenmeyer flask in a fume hood
     150 mL deionized water
     75 mL Glycerol
Stir until the two liquids are thoroughly mixed
Adjust the volume to 250 mL using deionized water
Sterile filter using a 0.2 um filter into a sterile 250 mL media bottle 
Store at 4o C until use
SECTION: References
9. 1.  Jainchill, J.L., S.A. Aaronson, and G.J. Todaro, Murine sarcoma and leukemia viruses: assay using clonal lines of contact-inhibited mouse cells. J Virol, 1969. 4(5): p. 549-53.
2.  Lowy, D.R., E. Rands, and E.M. Scolnick, Helper-independent transformation by unintegrated Harvey sarcoma virus DNA. J Virol, 1978. 26(2): p. 291-8.
3.  Miller, A.D. and C. Buttimore, Redesign of retrovirus packaging cell lines to avoid recombination leading to helper virus production. Mol Cell Biol, 1986. 6(8): p. 2895-902.
4.  Mann, R., R.C. Mulligan, and D. Baltimore, Construction of a retrovirus packaging mutant and its use to produce helper-free defective retrovirus. Cell, 1983. 33(1): p. 153-9.
5.  Danos, O. and R.C. Mulligan, Safe and efficient generation of recombinant retroviruses with amphotropic and ecotropic host ranges. Proc Natl Acad Sci U S A, 1988. 85(17): p. 6460-4.
6.  Daniel DiMaio - Yale University.  [Accessed February 3, 2024]; Available from: https://medicine.yale.edu/profile/daniel-dimaio/.
7.  Dwivedi, V. and D.J. Riese 2nd. Cell Culture Reagents and Sources - 2023-08-30. protocols.io 2023  [Accessed on February 3, 2024]; Available from: https://dx.doi.org/10.17504/protocols.io.261ged55wv47/v1.
8.  Falcon 352063 5 mL snap-cap tubes - VWR.  [Accessed February 3, 2024]; Available from: https://pr.vwr.com/store/product/4948460/falcon-round-bottom-high-clarity-polypropylene-tube-disposable-corning.