Generating Lentivirus-Infected Mouse Embryonic Stem Cells

Abstract

Source: Chiarella, A. M. et. al., Repressing Gene Transcription by Redirecting Cellular Machinery with Chemical Epigenetic Modifiers. J. Vis. Exp. (2018)

This video demonstrates the generation of genetically modified mouse embryonic stem cells using lentiviral transduction. Lentiviral particles deliver an antibiotic resistance marker, allowing stable genomic integration and selection of transduced cells.

Protocol

1. Infection of Mouse Embryonic Stem Cells (mESC) with Lentivirus

1. Grow low-passage (less than passage 35), feeder-free adapted chromatin in vivo assay (CiA) mESCs in high-glucose Dulbecco's Modified Eagle medium (DMEM) base media supplemented with 20% ESC-grade fetal bovine serum (FBS), 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 1x non-essential amino acids (NEAA), Pen/Strep, 2-mercaptoenthanol, and 1:500 leukemia inhibitor factor (LIF) in a 37 °C incubator with 5% CO₂.
   NOTE: The LIF is obtained from the supernatant of Lif-1C-alpha - producing Cos cells (COS LIF), which are collected in batch and frozen at -80 °C.
   1. Grow cells on plates that have been preincubated for 1 - 3 h with 0.1% gelatin in 1x phosphate-buffered saline (PBS), and subsequently washed with 1x PBS. Feed the cells daily and split them every 2 - 3 d, depending on their density.
      NOTE: Morphologically, embryonic stem cell (ES) colonies should be small, round, and distinct from each other.
2. Passage mESC into a 12-well plate format (100,000 cells/well) 1 d prior to infection.
   1. Count the cells with a hemocytometer. For cells grown in a 10-cm format, aspirate the old media, add 5 mL of 1x PBS, aspirate the PBS, and add 1 mL of 0.25% trypsin. Incubate the cells in trypsin for 8 min at 37 °C, rocking and tapping the cells halfway through the incubation. The goal of this step is to get the cells into a single-cell suspension.
3. 48 h after changing the media from the 293T human embryonic kidney (HEK) cells from the 15-cm plate, remove and transfer the supernatant to a 50-mL conical tube.
4. Centrifuge the supernatant at 300 x g for 5 min to pellet cell debris.
5. Filter the supernatant through a 0.45-µm membrane.
   NOTE: Make sure to use surfactant-free cellulose acetate (SFCA) membranes, as some other materials can retain the virus and significantly reduce yields.
6. Concentrate the virus with ultracentrifugation, using a centrifuge with an SW32 rotor, and spin it at 20,000 rpm (~72,000 x g) for 2.5 h at 4 °C.
7. While the virus concentrates under ultracentrifugation, treat the CiA mESCs in the 12-well plate with fresh ES media containing 5 µg/mL of polybrene.
8. When the virus concentration has finished, carefully aspirate the supernatant and suspend the virus pellet in 100 µL of 1x PBS (avoid excess bubbles).
9. Add the virus and 1x PBS to a 1.5-mL microfuge tube. Vortex/shake the tube at 300 x g (or at the lowest setting) to fully suspend the virus. Spin down the tube in a mini-tabletop centrifuge for 5 - 10 s to remove bubbles.
10. Add 30 µL of the virus to each well of a 12-well plate. Swirl the plate and then centrifuge the plate at 1,000 x g for 20 min.
    NOTE: The amount of virus added can be varied depending on the viral titer, which is inversely related to delivery construct size.
    1. Alternatively, flash-freeze the virus with liquid nitrogen and store it at -80 °C. However, freezing the virus will lower the viral titer.
11. Place the 12-well plate back in the 37 °C incubator and change the media the following morning (~16 h later) with fresh ES media.
12. After 48 h of infection, select for cells that integrated the viral plasmid by adding the appropriate antibiotic (i.e., 1.5 µg/mL of puromycin, 10 µg/mL of blasticidin, etc.). Change the media daily and wash the cells with 1x PBS if a majority of the cells are floating/dead. Keep the selection media on the cells for 72 - 96 h in a 37 °C incubator with 5% CO₂.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
DMEM	Corning	10-013CV
NEAA	Gibco	11140-050
HEPES (for tissue culture)	Corning	25-060-Cl
FBS	Atlantic Biologicals	S11550	Individual lots tested for quality
Virus centrifuge tubes	Beckman Coulter	344058
Virus filter membrane	Corning	431220
Polybrene	Santa Cruz Biotechnology	SC134220
0.25 % Trypsin	Gibco	25200-056	with EDTA
0.05 % Trypsin	Gibco	25400-054	with EDTA
Puromycin	InvivoGen	ant-pr
Ultracentrifuge	Optima	XPN-80
SW-32 centrifuge rotor	Beckman Coulter	14U4354
SW-32 Ti centrifuge buckets	Beckman Coulter	130.2
LentiX 293 Human embryonic kidney	Clontech	632180
PBS	Corning	46-013-CM
CiA:Oct4 mESC	The kind gift of G. Crabtree
Lif-1C-alpha - producing Cos cells	The kind gift of J. Wysocka