Establishing a Murine Tumor Cell Line In Vitro Using a Virus-Cas9 System

초록

Source: Prasad, M. et al. In Vitro Establishment of a Genetically Engineered Murine Head and Neck Cancer Cell Line using an Adeno-Associated Virus-Cas9 System. J. Vis. Exp. (2020)

This video demonstrates the in vitro transformation of murine epithelial cells through viral delivery of Cre recombinase and mutation-inducing components. It outlines the steps for Cas9 activation, cancer-gene modification, and the establishment of a tumorigenic murine cell line.

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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board. 

1. Adeno-associated virus (AAV) production

1. Day 1: Cell culture
   1. Seed 4 x 10⁶ Human embryonic kidney 293T (HEK293T) cells per 14.5 cm plate in 15 mL of Dulbecco's modified Eagle medium (DMEM). Prepare 10 plates for transfection with polyethylenimine (PEI) (1 µg/µL).
2. Day 2: Transfection of HEK293T cells using PEI
   1. Remove media and refeed with warm DMEM 1 h before the transfection.
   2. Prewarm transfection reagents and DMEM.
   3. Use 10 µg of the plasmid of interest containing AAV inverted terminal repeats (AAV pCM109 EFS Cre sg APC sg KRAS sg P53- KRAS HDR), 10 µg of the AAV 2/9n capsid plasmid (with the rep gene of AAV2 and the cap gene of AAV9), and 10 µg of the helper plasmid (pAdDelta F5 helper) per plate (see Table of Materials).
      NOTE: A new generation of helper plasmid - pAdDeltaF6 that can also be used for AAV production is available.
   4. Mix the plasmids in 1 mL of plain DMEM per plate. Then add 90 µL of polyethylenimine (total plasmid: PEI concentration = 1:3) to the plasmid mix and vortex briefly.
   5. Incubate the PEI-plasmid DNA mix for 20 min at room temperature (RT).
   6. Add the mix dropwise on top of the HEK293T cells and transfer the plates to the cell culture incubator at 37 °C with 5% CO₂ for 24 h.
3. Day 3: Medium change after transfection
   1. Remove the medium completely and add 15 mL of fresh DMEM.
4. Day 5: Harvesting the virus
   1. Prepare a dry ice/ethanol bath.
   2. Collect the cells with a cell scraper and transfer them into 50 mL tubes (2 plates per 50 mL tube).
   3. Spin tubes at 800 x g for 15 min at room temperature.
   4. Discard the supernatant from all the tubes. Add 0.5 mL of the lysis buffer (150 mM NaCl, 50 mM tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl), pH = 8.5) per plate (i.e., 5 mL for 10 plates) to the first tube only. Resuspend the cells and transfer the total volume to the next tube and continue until the last tube.
   5. Transfer the cell suspension to a fresh 50 mL tube. Wash the tubes with the same volume of lysis buffer (0.5 mL per plate) using the same transfer method as described in step 1.4.4.
   6. Subject the cell suspension to three rounds of ~10 min freeze/thaw cycles between a dry ice/ethanol bath and a 37 °C water bath. Vortex briefly after each thawing.
   7. If the purification is carried out on the same day, set the equilibration and elution buffer (see Table 1 for the recipe) at RT.
   8. Add 50 units of benzonase per plate and incubate at 37 °C for 1.5 h.
      NOTE: Benzonase is used to digest residual nucleic acids from the host producer cells and the plasmid DNA present in the cell suspension.
   9. Spin the tubes at 3,000 x g for 15 min at 4 °C.
   10. Collect the supernatant in a syringe using an 18 G steel needle and push the solution through a 0.45 µm filter into a 15 mL tube to obtain the crude lysate.
   11. Store the crude lysate at 4 °C for a few weeks until purification or continue purification.

2. AAV purification

1. Day 5 or later
   1. Place the equilibration and elution buffer at RT.
   2. Wash chromatography columns with 10 mL of the equilibration buffer.
   3. Add 0.5 mL per plate of heparin-agarose to the column and then add the equilibration buffer (4x the volume of the heparin-agarose). Mix the solutions by inverting the column and let the agarose sediment.
   4. Elute the equilibration buffer from the column by gravity.
      NOTE: Leave some equilibration buffer to prevent the agarose from drying out.
   5. Load the crude lysate onto the column and incubate for 2 h at 4 °C with constant agitation. Bring the column into an upright position and allow the agarose to sediment.
   6. Elute the crude lysate by gravity.
   7. Wash the column with the equilibration buffer (4x the volume of the heparin-agarose).
   8. Place a 100 kDa centrifugal protein filter below the column and elute the virus using an elution buffer (3x the volume of the heparin-agarose) into the filter.
      NOTE: The elution buffer should not exceed 15 mL.
   9. Centrifuge the filter at 3,000 x g for ~30 min until less than 1 mL is left in the filter.
   10. Fill up the filter with phosphate-buffered saline (PBS) and centrifuge at 3,000 x g for ~30 min until less than 1 mL is left in the filter. Repeat this step 2x to remove all salts.
   11. Concentrate the virus solution in the filter by centrifugation at 3,000 x g to arrive at a volume as small as possible (less than 200 µL).
   12. Aspirate the virus solution with a needle and syringe and push the solution through a 0.22 µm filter into a tube.
   13. Make aliquots of concentrated viral particles for storage.
       NOTE: The aliquots should be ~20 µL for short-term storage at 4 °C and ~5 µL for long-term storage at -80 °C.
   14. Determine the viral titer and viral transduction efficiency.

3. Isolation and culture of primary cells

1. Day 1
   1. Euthanize a 6-week-old male or female B6;129-Gt(ROSA)26Sor^{tm1(CAG-cas9*,-EGFP)Fezh}/J by CO₂ asphyxiation or any other IACUC approved protocol.
      NOTE: These clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 knockin mice have Cre recombinase-dependent expression of Cas9 endonuclease and enhanced green fluorescent protein (EGFP) directed by a CAG promoter. The upstream Lox-Stop-Lox (LSL) sequence present in the genome of these mice prevents the expression of Cas9 and EGFP in the absence of Cre recombinase. When used in combination with single guide RNAs (sgRNAs) and a Cre source, they allow editing of single or multiple mouse genes in vivo or ex vivo.
   2. Dissect the tongue from the euthanized mice using surgical scissors.
   3. Manually dissociate the tissue by mincing the tongue tissue into very small fragments using a scalpel. Collect the tissue fragments in a 15 mL tube containing 4.5 mL of Roswell Park Memorial Institute medium (RPMI) plain medium (without serum).
   4. Add the triple enzyme mix (200 µL) (see Table 1 for the recipe) to the tissue fragments.
   5. Incubate the tissue-enzyme mix at 37 °C for 30 min and tap the tube every 10 min to enhance enzymatic dissociation of the tissue.
   6. Add 5% fetal bovine serum (FBS) containing Hanks' balanced salt solution (HBSS)/PBS to the tissue-enzyme mix to stop the enzyme action.
   7. Filter the above cell suspension through a sterile 70 µm nylon mesh to separate the dispersed cells and larger tissue fragments.
   8. Wash the filtered cell suspension by centrifugation for 300 x g for 5 min in HBSS/PBS at RT.
   9. Resuspend the pellet in culture medium (10% FBS in RPMI/DMEM) and grow in 60 mm culture dishes until distinct cell colonies are formed.
      1. Culture cell aggregates are retained on top of the filter in a 60 mm culture dish containing 3 mL of complete media (10% FBS in RPMI/DMEM) until cell colonies are formed.
   10. Microscopically examine the primary cells for the presence of fibroblast contamination after 1 week of culture. Treat the primary cells developed from aggregates and cell suspensions with 0.25 trypsin 0.02% ethylenediaminetetraacetic acid (EDTA) solution at 37 °C for 1 min to remove fibroblasts.
       NOTE: Usually the primary culture from cell aggregates produces more colonies compared to single-cell suspensions. The cells from these colonies provide better transduction efficiency with AAV transduction.

4. AAV transduction of primary cells

1. Seed 2 x 10⁵ primary cells per well in 6-well plates in 2 mL of complete media (10% FBS in RPMI/DMEM).
2. The next day, transduce the cells with 10¹² viral genome/mL (10¹⁰ transducing units/mL) and incubate the cells in viral particle-containing media for 48 h at 37 °C.
3. Remove the viral particle-containing media and feed the AAV-transduced cells with complete media.
   NOTE: Only cells that underwent transduction will express green fluorescent protein (GFP) and start to proliferate. Cells that did not undergo transduction will eventually die within 2 weeks.

Table 1: Recipes of buffers used in this study

Cell lysis buffer for viral harvesting	Volume
150 mM NaCl	876.6mg
50 mM Tris-HCl	605.7mg
Use HCl to adjust pH 8.5
Molecular grade water	Make up to 100 mL
Equilibration buffer
1 mM MgCl2	9.52 mg
2.5 mM KCl	18.64mg
Mix all in PBS 1X and adjust pH to 7.2	Make up to 100 mL
Elution buffer
0.5 M NaCl	2.92g
Mix all in PBS 1X and adjust pH to 7.2	Make up to 100 mL
10X Triple Enzyme Stock Solution:
Collagenase	1 g final conc. [10 mg/ml]
Hyaluronidase	100 mg [1 mg/ml]
DNase	20,000 Units final conc. [200 mg/ml]
PBS 1X	Make up to 100 mL
Plasmid mix (for one 14.5cm Plate)
AAV pCM109 EFS Cre sg APC sg KRAS sg P53-KRAS HDR	10µg
AAV 2/9 capsid vector	10µg
pAD Delta F5 helper	10µg
PEI (1µg/µl Stock)	90µl
DMEM	1 mL

재료

이 논문에 사용된 재료 목록

이름	회사	카탈로그 번호	댓글
Cell lines
HEK93T	ATCC	CRL-3216
Culture Media, Chemicals and Reagents
DMEM	Biological Industries Israel Beit-Haemek Ltd.	01-055-1A
FBS	Biological Industries Israel Beit-Haemek Ltd.	04-127-1A
HBSS	Sigma	H6648
Heparin - Agarose	Sigma	H6508
NaCl	Bio Lab Ltd	1903059100
PBS	Biological Industries Israel Beit-Haemek Ltd.	02-023-1A
Tris buffer	MERCK Millipore	648311-1KG
PEI	Polysciences	23966-1
Enzymes
Benzonase	Sigma	E1014
Collagenase IV	Thermo Fisher Scientific	17104019
DNAse	Thermo Fisher Scientific	18047019
Hyaluronidase	MilliporeSigma	H3506
Trypsin	Biological Industries Israel Beit-Haemek Ltd.	03-050-1B
Mouse strains
B6;129-Gt(ROSA)26Sortm1(CAG-cas9*,-EGFP)Fezh/J	Jackson labs	24857
Plasmids
AAV pCM109 EFS Cre sg APC sg Kras sg P53- Kras HDR	Broad Institute of MIT		Kind gift from Dr Randall J Platt and Dr. Joseph Rosenbluh, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA
AAV 2/9 capsid vector	Addgene	112865
pAD Delta F5 helper	Ben Gurion University of the Negev		Provided by Dr Daniel Gitler, Department of Physiology and Cell Biology, Faculty of Health Sciences, and Zlotowski Center for Neuroscience, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel.
Plastic wares
Amicon-ULTRA filter 100 KDa	Millipore	UFC910024
0.22 µm sterile filters, 4 mm	Millex	SLGV004SL
0.45 µm sterile filters, 13 mm	Millex	SLHV013SL
Culture plates	Greiner Bio-One
Falcon tubes	Greiner Bio-One