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Method Article
A protocol is described for isolating stably transfected melanoma cell clones using glass cylinders. We focus on practical advice that may be decisive for the success of the entire procedure.
Cell populations that have stable changes in their genomic information are widely used by scientists as a research model. They do not require repeated cell transfection as it can lead to a heterogeneous cell population and variable transfection efficiency, affecting reproducibility. Moreover, they are preferable for large-scale analyses. The generation of stable cell clones is useful for a wide range of applications, such as research on gene functions and recombinant protein production. There are a few methods to obtain a homogenous cell population upon initial transient transfection. Here, we describe the isolation of single cell clones with glass cylinders. Although this method has been known for some time, there are a few crucial steps, and neglecting them may lead to failure. We have successfully used this method to obtain clones stably overexpressing a protein of interest (POI) or with knockout of a gene of interest (GOI). We describe preparation steps such as the optimization of selecting drug concentrations, preparation of glass cylinders, and validation of whether the obtained clones have the desired change in the expression of the GOI by PCR, western blot analysis, immunostaining, or gDNA sequencing (depending on the type of derived clones). We also discuss the phenotypic heterogeneity of well-established cell lines as this might be an issue in obtaining stable cell clones.
Stable transfection of mammalian cells is a routinely used method in several cell culture applications, including cancer research. Its advantage over transient transfection is that the introduced foreign genetic material cannot be lost due to environmental factors (e.g., cell confluency or replication stage) and cell division because it is integrated into the genome of the host1. Development of stably expressing cell lines can be laborious and challenging, but if a sustained expression of genes is required over an extended period, derivation of stable cell lines is a preferred option. The most common aim of transfection is to study the functions of a specific gene or gene product by overproduction or downregulation of its expression. However, the production of recombinant proteins and genetic therapies also requires the introduction of foreign genetic material into the cell2.
A clone is defined as a cell population derived from one individual cell. The isolation of single cell clones is a crucial aspect of cell biology when studying cells with genotypic and phenotypic variability. Three major techniques are used for deriving cell clones: the dilution technique, cloning ring technique3, and cell sorting technique4. Each one has advantages and disadvantages. We provide a detailed description of a method for isolating melanoma cell clones using the glass cylinder technique. The advantage of this method is that the cells exhibit moderate clonal growth from cell cultures with low density. Moreover, the cells selected have already demonstrated proliferation capability because they have already formed colonies5. This procedure involves seeding transfected cells sparsely, but not at limiting dilution, into a large vessel and allowing them to expand and form colonies for 2-3 weeks in the presence of a selective antibiotic. The individual colonies can then be isolated using glass cylinders that are placed over the colonies and adhered to the vessel using silicone grease. Next, cells are detached with trypsin and then transferred to multiwell plates for further culture in the presence of a selective medium.
There are a number of studies describing the isolation of clones, but we focus on showing details such as the size that clones should be after 2 weeks of selection, how to mark the location of the clones during their isolation, and how to choose an appropriate concentration of antibiotic for the selection. We focus on practical advice that may be decisive for the success of the entire procedure. The presented protocol allows us to obtain stable cell clones within 2-4 weeks. The method is easy and cheap and does not require complicated equipment. We share a technique that has allowed us to obtain many research models, including GSN KO clones6,7,8.
1. Cell subculture and seeding for drug concentration optimization
2. Optimization of selected drug concentration
3. Cell seeding and transfection
4. Generation of cell clones
5. Choosing the clones for isolation
6. Preparation of glass cylinders
7. Isolation of clones
8. Validation of the generated cell clones
Reagents | Volume |
DNA Polymerase Master Mix | 7.5 µl |
Forward primer (10 μM) | 1.5 µl |
Reverse primer (10 μM) | 1.5 µl |
Sterile water | 3.5 µl |
gDNA | 1 µl containing 10 ng gDNA |
Table 1. Composition of the PCR reaction mixture.
Steps | Temperature [°C] | Time [minutes: seconds] |
1 | 95 | 04:00 |
2 | 95 | 00:30 |
3 | 57 | 00:30 |
4 | 72 | 00:45 |
5 | go to 2, 35x | X |
6 | 72 | 10:00 |
7 | 8 | ∞ |
Table 2. Thermocycler settings for the PCR reaction.
Reagents | Volume |
5 x High-Fidelity DNA Polymerase Buffer | 4 µl |
dNTPs (10 mM) | 0.4 µl |
Forward primer (10 μM) | 1 µl |
Reverse primer (10 μM) | 1 µl |
Sterile water | 12.4 µl |
gDNA | 1 µl containing 10 ng gDNA |
High-Fidelity DNA Polymerase | 0.2 µl |
Table 3. Composition of the PCR reaction mixture.
Steps | Temperature [°C] | Time [minutes: seconds] |
1 | 98 | 00:30 |
2 | 98 | 00:30 |
3 | 61 | 00:30 |
4 | 72 | 01:00 |
5 | go to step 2, 35 times | X |
6 | 72 | 10:00 |
7 | 8 | ∞ |
Table 4. Thermocycler settings for the PCR reaction.
Reagents | Amount |
10 x buffer | 3 μl |
Restriction enzyme | 1 μl |
Sterile water | 20.7 μl |
Plasmid | 5.3 μl containing 5 μg of DNA |
Table 5. Composition of the restriction digestion reaction mixture.
9. Assess the cell line's phenotypic heterogeneity
Using the protocol presented, we provide a detailed demonstration of the isolation of stably transfected melanoma cell clones with the glass cylinder method (Figure 1A). Our studies relate to melanoma cell biology, and the most common research model that we use is the adherent and highly invasive A375 melanoma line. Since our research has shown that melanoma cells produce gelsolin (GSN) at a high level compared to other types of cancer14, we are investigating the role...
We have provided a detailed description of the isolation of stably transfected melanoma cell clones with glass cylinders. It is important to obtain control clones when deriving clones with changed expression of genes because the results obtained for the targeted modification clones should always be compared to results obtained for controls. In this way, we can check whether the transfection itself or culture with a selective antibiotic does not affect the results of the experiments, instead of some other factors, such as...
The authors declare no competing financial interests.
This work was supported by the National Center for Science, Poland (project #2016/22/E/NZ3/00654, granted to AJM).
Name | Company | Catalog Number | Comments |
15 ml centrifuge tube | GoogleLab Scientific | G66010522 | |
150 mm cell culturedish with 20 mm grid | Falcon | 353025 | |
24-wells plate | VWR International | 10062-896 | |
35 mm culture dish | Eppendorf | EP0030700112 | |
6-well plate | Eppendorf | EP0030700113 | |
96-well plate | VWR International | 10062-900 | |
Acetic acid, 80% solution | Chempur | 115687330 | |
Agarose | Prona-Abo | BLE500 | |
Antibiotic-Antimycotic | Gibco | 15240062 | |
anti-GAPDH antibodies | Santa Cruz Biotechnology Inc., | sc-47724 | |
anti-GSN antibodies - clone C20 | Santa Cruz Biotechnology Inc., | sc-6405 | |
anti-GSN antibodies - clone GS-2C4 | Sigma-Aldrich | G44896 | |
Bovine Serum Albumin (BSA) | Sigma-Aldrich | A3294 | |
Color Taq PCR Master Mix (2x) | EurX | E2525 | |
Control Double Nickase Plasmid | Santa Cruz Biotechnology Inc., | sc-437281 | |
Coverslips | bionovo | 16283 | |
Dako Mounting medium | Clontech | S3023 | |
DMSO - Dimethyl sulfoxide | applichem | A3672,0250 | |
DNA Purification Kit | EurX | 3555-02 | |
donkey anti- mouse-Alexa Fluor 488 | Invitrogen | # A-21202 | |
DTT - 1,4-Dithiothreitol | Sigma-Aldrich | 10197777001 | |
EcoRI | Thermo Fisher Scientific | FD0274 | |
EDTA- ethylenediaminetetraacetic acid | Poch (Pol-Aura) | 593280117 | |
EGTA - ethylene glycol-bis(2-aminoethyl ether)- N,N,N’,N’-tetraacetic acid | Sigma-Aldrich | E0396 | |
FBS - Fetal Bovine Serum | Gibco | 10270-106 | |
Gelsolin CRISPR Plasmids | Santa Cruz Biotechnology Inc., | sc-401005-NIC | |
Formaldehyde | Sigma-Aldrich | P6148 | |
Glycerol | Sigma-Aldrich | L-4909 | |
high glucose Dulbecco’s modified Eagle’s medium with reduced concentration (1.5 g/l) of NaHCO3 | Polish Academy of Science,Wroc![]() | 11-500 | |
Hoechst 33342 | Thermo Fisher Scientific | H3570 | |
L-Glutamine | Gibco | 25030-024 | |
Lignin | Bionovo | B-0521 | |
Lipofectamine 3000 Transfection Reagent | Invitrogen | L3000-008 | |
Na3VO4 | Sigma-Aldrich | S6508 | |
Na4P2O7 | Sigma-Aldrich | P8010 | |
NaF | Sigma-Aldrich | 450022 | |
NEBuilder Assembly Tool | http://nebuilder.neb.com/#!/ | ||
pACGFP-C1 | Clontech | ||
PageRuler Prestained Protein Ladder | Thermo Fisher Scientific | 26616 | |
Perfect 100 bp DNA ladder | EurX | E3134 | |
phalloidin-Alexa Fluor 568 | Invitrogen | A12380 | |
Phosphatase Inhibitor Cocktail 2 | Sigma-Aldrich | P5726 | |
Phosphatase Inhibitor Cocktail 3 | Sigma-Aldrich | P0044 | |
Phusion High-Fidelity DNA Polymerase | Thermo Fisher Scientific | F530S | |
Pierce BCA Protein Assay Kit | Thermo Fisher Scientific | 23225 | |
polyethylenimine (PEI) | Sigma-Aldrich | 408727 | |
protease inhibitor cocktail | Sigma-Aldrich | P8340 | |
puromycin | InviviGen | ant-pr | |
SDS - sodium dodecyl sulfate | Sigma-Aldrich | L4509 | |
silicone | CX80 Polska | ||
Sodium chloride - NaCl | Chempur | 7647-14-5 | |
sodium deoxycholate | Sigma-Aldrich | D6750259 | |
sucrose | Poch (Pol-Aura) | PA-06-772090110 | |
the glass cylinders | Sigma-Aldrich | C3983-50EA | |
Tissue Culture Flask 25mL | VWR International | 10062-868 | |
Tissue-culture 75 cm2 flask | VWR | 10062-872 | |
Trisma base | Sigma-Aldrich | T1503 | |
Triton X-100 | Sigma-Aldrich | X100 | |
trypsin | Polish Academy of Science,Wroc![]() | 20-500 | |
urea | Sigma-Aldrich | 8656 | |
xylene solution | Chempur | 115208603 |
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