Protocol for G418 Selection of CRISPR/Cas9-Edited Cells
Protocol for G418 Selection of CRISPR/Cas9-Edited Cells
1 Overview
1.1 Purpose and Scope
This protocol is used to establish a routine workflow for G418 selection, single-clone isolation, clonal expansion, and stable cell line maintenance after CRISPR/Cas9 editing of mammalian cells. It is applicable to CRISPR/Cas9 vectors or co-transfection systems carrying the neomycin resistance gene (neo). This method is mainly used to enrich cell populations that have successfully received the selection vector and to provide a basis for subsequent single-clone establishment and target-site validation.
1.2 Method Principle
(1) G418 is an aminoglycoside antibiotic that inhibits protein synthesis in eukaryotic cells.
(2) When cells express aminoglycoside 3'-phosphotransferase encoded by the neo gene, G418 can be inactivated, allowing cells to survive under selection pressure.
(3) Cells that fail to receive the vector or do not effectively express neo gradually die in the presence of G418. Therefore, G418 can be used to enrich transfection-positive cells.
(4) G418 selection is essentially a selection method for cells positive for vector introduction, rather than a direct determination of cells with correct target editing. Therefore, subsequent PCR and sequencing validation of the target site are still required.
1.3 Key Points in Experimental Design
(1) The G418 selection concentration is strongly cell-line specific, and a kill curve must be established before formal experiments.
(2) Cells should be actively proliferating when selection begins. The recommended starting confluency is 20%–25%.
(3) A recovery period should be included after transfection to allow sufficient expression of Cas9, sgRNA, and neo protein.
(4) During formal selection, cells should be passaged at low density to allow resistant clones to form and be isolated.
2 Reference G418 Concentrations for Common Cell Lines
Table 1 Reference G418 selection and maintenance concentrations for common mammalian cell lines
Cell Line | Recommended Selection Concentration (μg/mL) | Recommended Maintenance Concentration (μg/mL) |
HEK293 | 200–500 | 100–250 |
CHO | 400–1000 | 200–500 |
HeLa | 400–800 | 200–400 |
NIH3T3 | 400–500 | 200–250 |
Jurkat | ~1000 | ~500 |
NRK | 200 | 100 |
P19 | 400 | 200 |
The concentrations above should be used only as starting ranges for preliminary experiments. The final working concentration should be determined based on the kill curve results obtained in each laboratory.
3 Materials and Reagents
3.1 Main Materials
(1) Target mammalian cell line.
(2) CRISPR/Cas9 plasmid containing a neo selection element, or the corresponding co-transfection plasmid system.
(3) 24-well plates, 6-well plates, culture dishes, and culture flasks.
(4) Cloning rings or sterile pipette tips.
(5) Hemocytometer or automated cell counter.
(6) Inverted microscope.
3.2 Main Reagents
(1) Complete culture medium.
(2) G418 stock solution or working solution.
(3) CRISPR/Cas9 plasmid containing a neo selection element, or the corresponding co-transfection plasmid system.
(4) Transfection system: lipid-based transfection, electroporation, or viral delivery system selected according to cell type.
(5) PBS.
(6) Trypsin or cell dissociation solution.
(7) Trypan blue staining solution.
(8) Genomic DNA extraction reagent.
(9) PCR amplification reagents.
(10) Sequencing-related reagents or sequencing service.
4 Establishment of a G418 Kill Curve
4.1 Purpose
The kill curve is used to determine the lowest G418 concentration that kills all non-transfected cells within 7–10 d. This concentration is used as the selection concentration for subsequent formal selection.
4.2 Cell Seeding
(1) Seed the target cells in a 24-well plate.
(2) Control the cell confluency on the following day to approximately 20%–25%.
(3) Set replicate wells for each concentration and include a no-G418 control well.
4.3 G418 Gradient Setup
On the following day, prepare a G418 concentration gradient using complete culture medium. Common starting concentrations include 0, 100, 200, 400, 600, 800, 1000, and 1200 μg/mL. The range may be further refined according to the characteristics of the cell line.
4.4 Drug Addition and Observation
(1) Remove the original culture medium and add G418 selection medium at the corresponding concentration.
(2) Observe cell status under the microscope daily and record cytotoxic changes such as cell rounding, detachment, and lysis.
(3) Replace with fresh medium containing the same concentration of G418 every 2–3 d.
4.5 Endpoint Determination
After 7–10 d of culture, determine the lowest G418 concentration that kills all non-transfected cells. This concentration is used as the formal selection concentration.
5 Formal G418 Selection of CRISPR/Cas9-Edited Cells
5.1 Transfection
Introduce the CRISPR/Cas9-neo plasmid or co-transfection system according to the selected transfection method. For lipid-based transfection, optimize DNA amount, transfection reagent ratio, and cell density. For electroporation, optimize voltage, pulse duration, and buffer system. For viral delivery, define the multiplicity of infection and infection duration.
5.2 Recovery Period
After transfection, culture cells in complete medium without G418 for 48–72 h to allow cell recovery and expression of Cas9, sgRNA, and neo protein.
5.3 Initiation of Selection
(1) After the recovery period, passage the cells at low density into a new culture vessel.
(2) Common passaging ratios are 1:10 or 1:20 to provide sufficient space for subsequent resistant clone growth.
(3) Add culture medium containing the pre-determined optimal G418 selection concentration.
5.4 Maintenance of Selection
(1) Replace with fresh G418-containing medium every 3–4 d.
(2) Continuously monitor the death of non-transfected cells and the formation of resistant clones.
(3) In most cell lines, well-defined resistant clones can appear within 1–3 weeks.
6 Single-Clone Isolation and Expansion
6.1 Timing of Clone Picking
When clear, separated, and appropriately sized resistant clones appear in the culture dish, single-clone isolation can be performed. If clones are too dense, the seeding density or selection conditions should be re-optimized.
6.2 Isolation Methods
(1) Cloning rings can be used with local digestion to isolate single clones.
(2) A sterile pipette tip can also be used to manually pick individual clones.
(3) Transfer each clone separately into a new 24-well plate for expansion culture.
(4) Maintain G418 selection pressure during the expansion stage.
6.3 Expansion Culture
After single clones resume growth, gradually expand the culture volume while recording the clone number, picking date, and culture status.
7 Validation of Editing Results
7.1 Molecular Validation
After single clones have expanded to a sufficient number, extract genomic DNA and perform PCR amplification. Confirm whether the expected edit has occurred at the target site using Sanger sequencing or other molecular methods.
7.2 Maintenance of Stable Cell Lines
After correct editing is confirmed, stable cell lines can be maintained in a lower concentration of G418. The maintenance concentration is usually approximately 50% of the formal selection concentration.
8 Key Points for Result Interpretation
8.1 Kill Curve
(1) The no-G418 control wells should maintain normal growth.
(2) As the G418 concentration increases, the rate of cell death should gradually increase.
(3) The optimal selection concentration should meet two requirements: complete killing of non-transfected cells and survival and expansion of resistant cells.
8.2 Formal Selection
(1) If all cells die rapidly after drug addition and no clones form, the G418 concentration may be too high, the recovery period may be insufficient, or transfection efficiency may be too low.
(2) If a large number of non-transfected cells survive for an extended period, the G418 concentration may be too low or the medium replacement frequency may be insufficient.
(3) If resistant clones form but cannot be stably expanded, evaluate intrinsic cell tolerance, neo expression level, and whether selection pressure is too strong.
9 Critical Control Points
9.1 Cell Status
G418 selection is suitable for actively dividing cells. Poor cell status, overconfluency, or contamination can markedly reduce selection efficiency.
9.2 Vector Design
If the CRISPR/Cas9 elements and neo are not in the same expression system, co-transfection efficiency and co-expression ratio should be carefully considered. Otherwise, selection-positive but editing-negative cells may occur.
9.3 Recovery Period
A recovery period that is too short may result in insufficient neo expression, whereas a recovery period that is too long may increase the background of non-transfected cells.
9.4 Maintenance of Selection Pressure
Delayed medium replacement can reduce selection pressure and affect the purity of resistant clones.
10 Common Problems and Cause Analysis
10.1 Unclear Kill Curve
(1) Cell seeding density is too high.
(2) G418 concentration gradient is too narrow.
(3) Medium replacement is not timely.
(4) Observation period is insufficient.
10.2 No Resistant Clones Form
(1) Low transfection efficiency.
(2) Insufficient recovery period.
(3) G418 concentration is too high.
(4) Cells have poor tolerance to exogenous gene expression.
10.3 High Number of False Positives
(1) G418 concentration is too low.
(2) Formal selection begins too late.
(3) Selection medium replacement frequency is too low.
10.4 Mixed Single Clones
(1) Seeding density is too high.
(2) Clone picking is performed too late.
(3) Clone picking is incomplete.
11 Quality Control
11.1 Preliminary Experiment Control
(1) A G418 kill curve should be re-established for each new cell line.
(2) After changing the culture system, serum batch, or culture conditions, the selection concentration should be revalidated when necessary.
11.2 Formal Selection Control
(1) Include a non-transfected drug-treated control.
(2) Include a transfected no-drug control.
(3) If necessary, include an empty-vector neo control to distinguish the selection effect from the editing effect.
11.3 Clone Validation Control
(1) Each single clone should be independently numbered.
(2) A cryopreserved backup should preferably be retained before sequencing.
(3) G418 selection positivity and target editing positivity should be interpreted separately.
12 Safety and Operating Standards
12.1 Personal Protection
(1) Wear a lab coat and disposable gloves during the experiment.
(2) All cell culture, transfection, and drug addition steps should be performed under sterile conditions.
12.2 Waste Disposal
(1) G418-containing culture medium waste should be collected separately.
(2) Cell culture consumables, pipette tips, and culture plates should be disposed of according to biological waste handling standards.
(3) PCR and sequencing samples should be processed separately from the cell culture area to avoid cross-contamination.
13 Related Reagent and Material Selection
Table 2 Reagent and material selection for G418 selection of CRISPR/Cas9-edited cells
Cat. No. | Product Name | Grade and Specification | Corresponding Step | Use |
HEK 293 Medium, with Glutamine | BioReagent,for cell culture,sterile-filtered | Complete medium | Suitable for recovery culture, selection culture, and clonal expansion of HEK293 and related cells | |
DMEM, High Glucose | sterile-filtered, BioReagent, endotoxin tested, for cell culture, sterile | Complete medium | Suitable for routine culture and G418 selection of various adherent cells | |
Ham's F-12 Nutrient Mixture | sterile-filtered, BioReagent, endotoxin tested, for cell culture | Complete medium | Can be used for CHO and related cell culture systems | |
RPMI 1640 Medium | sterile-filtered, BioReagent, endotoxin tested, for cell culture | Complete medium | Suitable for selection culture of suspension cells such as Jurkat | |
G418 Solution | 25mg/ml,sterile | G418 selection | Suitable for kill curve establishment and formal selection | |
G418 Sulfate Solution (50 mg/mL, Sterile) | sterile-filtered,BioReagent,for cell culture,50 mg/ml | G418 selection | Suitable for preparing higher-concentration stock solutions and maintaining selection | |
293 Cell-Specific Transfection Reagent | BioReagent,for cell culture,Suitable for molecular biology,sterile | Transfection | Suitable for plasmid transfection of HEK293 and related cells | |
Common Cell Transfection Reagent | BioReagent,for cell culture,Suitable for molecular biology,sterile | Transfection | Suitable for CRISPR plasmid delivery into routine adherent cells | |
Universal Cell Transfection Reagent | BioReagent,for cell culture,Suitable for molecular biology,sterile | Transfection | Suitable as a general transfection system for various cell types | |
Lipo2000 Transfection Reagent |
| Transfection | Suitable for routine plasmid transfection system establishment | |
Branched PEI Transfection Reagent (MW 25000) | BioReagent,ready-to-use,for cell culture,Suitable for molecular biology,sterile | Transfection | Suitable for plasmid transfection and some viral packaging systems | |
Linear Polyethylenimine Hydrochloride (MW 40,000), Transfection Grade | BioReagent, for cell culture, Suitable for molecular biology, Transfection Grade, Linear,Mw 4,000 | Transfection | Can be used as an alternative PEI-based transfection system | |
Calcium Phosphate Cell Transfection Kit | BioReagent,sterile,Suitable for molecular biology | Transfection | Suitable for plasmid delivery by calcium phosphate transfection | |
Lentivirus Packaging Transfection Reagent | BioReagent,for cell culture,Suitable for molecular biology,sterile | Viral delivery | Suitable for CRISPR lentiviral packaging systems | |
Viral Transfection Enhancer | BioReagent,for cell culture,Suitable for molecular biology,sterile,ready-to-use | Viral delivery | Suitable for enhancing viral infection efficiency | |
PBS (pH 7.4, Sterile) | BioReagent,Low Endotoxin,sterile-filtered,for cell culture | Washing | Suitable for cell washing and local processing | |
PhosphateBuffered Saline（PBS）1X concentrate | 1X,sterile,pH7.2-7.4 | Washing | Can be used as a ready-to-use PBS alternative | |
PBS | 1 L/pouch | Washing buffer preparation | Suitable for batch preparation of PBS | |
Trypsin Solution | BioReagent, for cell culture, sterile | Cell passaging/clonal expansion | Used for reseeding before selection, clonal expansion, and passaging | |
Trypan Blue Dyeing Kit | 0.40% | Cell counting/viability assessment | Used for viability detection during kill curve establishment and selection | |
Blood/Cell/Tissue Genomic DNA Extraction Kit (Spin Column) | BioReagent | Genomic DNA extraction | Suitable for DNA extraction after single-clone expansion | |
Magnetic Universal Genomic DNA Kit | BioReagent, for DNA and RNA applications | Genomic DNA extraction | Can be used as a general DNA extraction alternative | |
Taq-Plus PCR Master Mix (2x) | 2× | PCR amplification | Suitable for routine amplification validation of target sites | |
Taq-Plus PCR Forest Mix (2x) | 2× | PCR amplification | Suitable for PCR amplification and pre-electrophoresis processing | |
HiFi Hotstart PCR Mix | BioReagent,DNase, RNase free,PCR Reagent,UltraBio™,Suitable for molecular biology,for DNA and RNA applications,25μL/T | PCR amplification | Suitable for amplification validation requiring higher specificity and accuracy | |
2×Flash PCR MasterMix (Dye) |
| PCR amplification | Suitable for routine rapid PCR detection |