Isolation of Stably Transfected Melanoma Cell Clones

Summary

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.

Abstract

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.

Introduction

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 clones^6,7,8.

Protocol

1. Cell subculture and seeding for drug concentration optimization Warm the cell culture medium (in this case, Dulbecco's modified Eagle's medium with a reduced concentration (1.5 g/L) of NaHCO3, 10% (v/v) fetal bovine serum (FBS), 1% (v/v) L-glutamine, and 1% (v/v) antibiotic-antimycotic) and trypsin solution in a water bath to 37 °C. Aspirate and discard the cell culture medium from the A735 cells growing in a T25 cell culture flask. The cell line is obtain…

Representative Results

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…

Discussion

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…

Materials

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,Wrocaw, Poland	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,Wrocaw, Poland	20-500
urea	Sigma-Aldrich	8656
xylene solution	Chempur	115208603