Production of Retroviral Particles for Gene Transfer

Abstract

Source: Zjablovskaja, P., et al., Proliferation and Differentiation of Murine Myeloid Precursor 32D/G-CSF-R Cells. J. Vis. Exp. (2018)

This video demonstrates the production of recombinant retroviral particles by transfecting human kidney-derived packaging cells, enabling stable gene delivery to target cells for downstream applications such as genetic modification, functional assays, or lineage differentiation studies.

Protocol

1. Production of retrovirus
   1. Plate a single cell suspension of Bosc23 cells (6 × 10⁶) in a 16 cm Petri dish and cultivate in 18 mL of DMEM (Dulbecco's Modified Eagle Medium) containing 10% FBS (fetal bovine serum) until the confluency of the culture reaches 80 % (24 h).
      NOTE: Cells should grow in a monolayer and not form clumps in culture. Cell counting can be performed using different methods (valid for the rest of the protocols presented here). In case of a low number of samples, manual counting under the microscope using the Bürker chamber is recommended. In this case, use Trypan blue for exclusion of dead cells. Mix cells 1:1 with 0.4% Trypan Blue in PBS (phosphate-buffered saline). To perform the counting of a large number of samples, an automatic cell counter can be employed.
   2. Combine 40 µg of retroviral construct (such as MSCV), 20 µg of pCL-Eco (packaging vector), 80 µl of PEI (polyethylenimine), and 2 mL of reduced-serum medium (e.g., Opti-MEM (Minimum Essential Medium) (without antibiotics). Incubate this mixture for 20 min at room temperature (RT).
   3. Carefully replace the Bosc23 cells medium with 16 mL DMEM supplemented with 2% FBS. Pre-warm the medium to 37 °C before use.
      NOTE: Do not use antibiotics during transfection, since they may reduce transfection efficiency.
   4. Add the mixture prepared in 1.2. dropwise and carefully into the Bosc23 culture, and incubate for 4 h at 37 °C. Limit incubation to less than 6 h due to PEI toxicity.
   5. After incubation, change the Bosc23 medium to 18 mL pre-warmed DMEM containing 10% FBS, and cultivate cells for 48 h at 37 °C. Place dishes in a biosafety level 2 laboratory, and follow standard safety procedures.
   6. Collect Bosc23 medium (containing ecotropic retroviral particles) using a 25-mL serological pipette into a 50-mL conical tube, and store at 4 °C.
      NOTE: (Important) Transfection efficiency should reach 90% to produce a high-titer virus.
   7. Add 18 mL pre-warmed DMEM 10% FBS to Bosc23 cells and cultivate for 24 h.
   8. Repeat step 1.6.
      NOTE: Retroviral supernatants from 1.6 and 1.8 can be pooled once they reach the same temperature (4 °C) to preserve viral integrity.
   9. To avoid Bosc23 contamination in viral supernatants, spin collected virus at 1500 × g for 10 min at 4 °C. Aliquot virus, snap freeze using dry-ice or liquid nitrogen, and store at -80 °C. However, note that freshly prepared virus is of higher quality in terms of infection efficiency than frozen stocks.
      NOTE: (Important) Avoid repetitive freezing/thawing of the virus, since it leads to viral degradation.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
DMEM (Dulbecco's Modified Eagle Medium)	Thermo Fisher Scientific, Waltham, MA, USA	15028
Opti-MEM ((Minimum Essential Medium) - Reduced Serum Medium	Thermo Fisher Scientific, Waltham, MA, USA	31985-047	L-Glutamine, Phenol Red
Fetal bovine serum (FBS)	Thermo Fisher Scientific, Waltham, MA, USA	10270	Used for culturing HEK293T, NIH3T3, BOSC23 cells
Penicillin	Sigma-Aldrich (Merck, Kenilworth, NJ, USA)	P3032
Streptomycin	Sigma-Aldrich (Merck, Kenilworth, NJ, USA)	S9137	Streptomycin sulfate salt powder
Gentamicin	Sigma-Aldrich (Merck, Kenilworth, NJ, USA)	G1914
Polyethylenimine	Polyscience, Warrington, PA, USA	23966	Linear, MW 25,000 (PEI 25000)