A subscription to JoVE is required to view this content. Sign in or start your free trial.
Many upregulated genes stimulate tumor cell migration and invasion, leading to poor prognosis. Determining which genes regulate tumor cell migration and invasion is critical. This protocol presents a method for investigating the effects of the increased expression of a gene on the migration and invasion of tumor cells in real time.
Tumor cells are highly motile and invasive and display altered gene expression patterns. Knowledge of how changes in gene expression regulate tumor cell migration and invasion is essential for understanding the mechanisms of tumor cell infiltration into neighboring healthy tissues and metastasis. Previously, it was demonstrated that gene knockdown followed by the impedance-based real-time measurement of tumor cell migration and invasion enables the identification of the genes required for tumor cell migration and invasion. Recently, the mRNA vaccines against SARS-CoV-2 have increased interest in using synthetic mRNA for therapeutic purposes. Here, the method using synthetic mRNA was revised to study the effect of gene overexpression on tumor cell migration and invasion. This study demonstrates that elevated gene expression with synthetic mRNA transfection followed by impedance-based real-time measurement may help identify the genes that stimulate tumor cell migration and invasion. This method paper provides important details on the procedures for examining the effect of altered gene expression on tumor cell migration and invasion.
Tumor cell motility plays a crucial role in metastasis1,2. The spread of tumor cells to neighboring and remote healthy tissues makes cancer treatment difficult and contributes to recurrence3,4. Therefore, it is essential to understand the mechanisms of tumor cell motility and develop relevant therapeutic strategies. Since many tumor cells have altered gene expression profiles, it is crucial to understand which changes in the gene expression profile lead to altered tumor cell motility5,6.
1. Synthesis of mRNA
NOTE: For the mRNA synthesis, all the reagents and equipment must be specially treated to inactivate the RNases before use. See the Table of Materials for details about all the materials, instruments, and reagents used in this protocol.
Crk and CrkL proteins play important roles in the motility of many cell types, including neurons22, T cells23, fibroblasts18,19, and a variety of tumor cells13. Since Crk and CrkL proteins have been reported to be elevated in glioblastoma24,25,26, the effects of the overexpression of CrkI, a splice v.......
Migration and invasion are important features of tumor cells. Measuring the motility of tumor cells and understanding the underlying mechanism that controls tumor cell motility provide critical insights into therapeutic interventions2,27. Several methods have been developed to study cell migration7. The wound-healing assay using scratches or culture inserts is a simple and frequently used method that provides contrasting images of gap clos.......
The authors thank the Medical Writing Center at Children's Mercy Kansas City for editing this manuscript. This work was supported by Natalie's A.R.T. Foundation (to T.P.) and by an MCA Partners Advisory Board grant from Children's Mercy Hospital (CMH) and the University of Kansas Cancer Center (KUCC) (to T.P.).
....Name | Company | Catalog Number | Comments |
AlphaImager HP | ProteinSimple | 92-13823-00 | Agarose gel imaging system |
α-Tubulin antibody | Sigma | T9026 | Used to detect α-tubulin protein (dilution 1:3,000) |
CIM-plate 16 | Agilent Technologies, Inc | 5665825001 | Cell invasion and migration plates |
Crk antibody | BD Biosciences | 610035 | Used to detect CrkI and CrkII proteins (dilution 1:1,500) |
CrkL antibody | Santa Cruz | sc-319 | Used to detect CrkL protein (dilution 1:1,500) |
Dulbecco’s Modified Eagle’s Medium (DMEM) | ATCC | 302002 | Cell culture medium |
Dulbecco's phosphate-buffered saline (DPBS) | Corning | 21-031-CV | Buffer used to wash cells |
Fetal bovine serum (FBS) | Hyclone | SH30910.03 | Culture medium supplement |
Heracell VIOS 160i CO2 incubator | Thermo Scientific | 51030285 | CO2 incubator |
IRDye 800CW goat anti-mouse IgG secondary antibody | Li-Cor | 926-32210 | Secondary antibody for Western blot analysis (dilution 1:10,000) |
IRDye 800CW goat anti-rabbit IgG secondary antibody | Li-Cor | 926-32211 | Secondary antibody for Western blot analysis (dilution 1:10,000) |
Lithium chloride | Invitrogen | AM9480 | Used for RNA precipitation |
Matrigel matrix | Corning | 354234 | Extracellular matrix (ECM) gel |
MEGAscript T7 transcription kit | Invitrogen | AM1334 | Used for RNA synthesis |
Millennium RNA markers | Invitrogen | AM7150 | Used for formaldehyde agarose gel electrophoresis |
Mini centrifuge | ISC BioExpress | C1301P-ISC | Used to spin down cells |
Mouse brain QUICK-Clone cDNA | TaKaRa | 637301 | Source of genes (inserts) for cloning |
NanoQuant | Tecan | M200PRO | Nucleic acid quantification system |
Neon electroporation system | ThermoFisher Scientific | MPK5000 | Electroporation system1 |
Neon transfection system 10 µL kit | ThermoFisher Scientific | MPK1025 | Electroporation kit |
Neon transfection system 100 µL kit | ThermoFisher Scientific | MPK10096 | Electroporation kit |
NorthernMax denaturing gel buffer | Invitrogen | AM8676 | Used for formaldehyde agarose gel electrophoresis |
NorthernMax formaldehyde load dye | Invitrogen | AM8552 | Used for formaldehyde agarose gel electrophoresis |
NorthernMax running buffer | Invitrogen | AM8671 | Used for formaldehyde agarose gel electrophoresis |
Nuclease-free water | Teknova | W3331 | Used for various reactions during mRNA synthesis |
Odyssey CLx Imager | Li-Cor | Imager for Western blot analysis | |
pcDNA3.1/myc-His | Invitrogen | V80020 | The vector into which inserts (mouse CrkI and CrkL cDNAs) were cloned |
pFLAG-CMV-5a | Millipore Sigma | E7523 | Source of the FLAG epitope tag |
Phenol:chloroform:isoamyl alcohol | Sigma | P2069 | Used for DNA extraction |
PmeI | New England BioLabs | R0560L | Used to linearize the plasmids for mRNA synthesis |
Poly(A) tailing kit | Invitrogen | AM1350 | Used for poly(A) tail reaction |
Polystyrene tissue culture dish (100 x 20 mm style) | Corning | 353003 | Used for culturing cells before transfection |
Polystyrene tissue culture dish (35 x 10 mm style) | Corning | 353001 | Used for culturing transfected cells |
Proteinase K | Invitrogen | 25530049 | Used to remove protein in the reaction mixture |
Purifier Axiom Class II, Type C1 | Labconco Corporation | 304410001 | Biosafety cabinet for sterile handling of cells |
Resuspension Buffer R | ThermoFisher Scientific | A buffer included in the electroporation kits, MPK1025 and MPK10096. The buffer is used to resupend cells before electroporation, and its composition is proprietary information. | |
RNaseZap | Invitrogen | AM9780 | RNA decontamination solution |
Scepter | Millipore | C85360 | Handheld automated cell counter |
ScriptCap 2'-O-methyltransferase kit | Cellscript | C-SCMT0625 | Used for capping reaction |
ScriptCap m7G capping system | Cellscript | C-SCCE0625 | Used for capping reaction |
Sodium dodecyl sulfate solution | Invitrogen | 15553-035 | Detergent used for the proteinase K reaction |
Sorvall Legend XT centrifuge | Thermo Scientific | 75004532 | Benchtop centrifuge to spin down cells |
Trypsin-EDTA | Gibco | 25300-054 | Used for dissociation of cells |
U-118MG | ATCC | HTB15 | An adherent cell line derived from a human glioblastoma patient |
Vinculin antibody | Sigma | V9131 | Used to detect vinculin protein (dilution 1:100,000) |
xCELLigence RTCA DP | Agilent Technologies, Inc | 380601050 | Instrument used for real-time cell analysis |
1Electroporation parameters and other related information for various cell lines are available on the manufacturer's homepage (https://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/neon-transfection-system/neon-transfection-system-cell-line-data.html?). |
This article has been published
Video Coming Soon
ABOUT JoVE
Copyright © 2024 MyJoVE Corporation. All rights reserved