Establishing Cell Lines Overexpressing DR3 to Assess the Apoptotic Response to Anti-mitotic Therapeutics

Summary

Establishing a stable cell line overexpressing a gene of interest to study gene function can be done by stable transfection-picking single clones after transfecting them via retroviral infection. Here we show that HT29-DR3 cell lines generated in this way elucidate the mechanisms by which death receptor 3 (DR3) contributes to antimitotics-induced apoptosis.

Abstract

Studying the function of a gene of interest can be achieved by manipulating its level of expression, such as decreasing its expression with knockdown cell lines or increasing its expression with overexpression cell lines. Transient and stable transfection are two methods that are often used for exogenous gene expression. Transient transfection is only useful for short-term expression, whereas stable transfection allows exogenous genes to be integrated into the host cell genome where it will be continuously expressed. As a result, stable transfection is usually employed for research into long-term genetic regulation. Here we describe a simple protocol to generate a stable cell line overexpressing tagged death receptor 3 (DR3) to explore DR3 function. We picked single clones after a retroviral infection in order to maintain the homogeneity and purity of the stable cell lines. The stable cell lines generated using this protocol render DR3-deficient HT29 cells sensitive to antimitotic drugs, thus reconstituting the apoptotic response in HT29 cells. Moreover, the FLAG tag on DR3 compensates for the unavailability of good DR3 antibody and facilitates the biochemical study of the molecular mechanism by which antimitotic agents induce apoptosis.

Introduction

Heterologous gene expression is often employed to study the function of genes of interest. Two methods, transient and stable transfection, are prevalently used in cells and molecular biology to insert a segment of DNA or RNA into a host cell^1,2. Transiently-transfected DNA or RNA cannot be passed on to daughter cells, so the genetic alteration can only be retained for a short period of time. On the other hand, in stably transfected cells, exogenous genes are integrated into the host cell genome, sustaining its expression in the cell line. Thus, stable transfection is a method that is usually reserved for resea....

Protocol

Please note that all the mouse experiments described here were approved and performed in accordance with the Institutional Animal Care and Use Committee (IACUC) at Tsinghua University.

1. Generation of DR3 Overexpression Cell Lines

1. Construct the plasmid for DR3 expression (pMXs-IRES-DR3-FLAG) by inserting full-length DR3 cDNA with 3x FLAG at the C-terminus into retroviral vector pMXs-IRES-Blasticidin at the restriction sites of BamHI/XhoI.
2. Grow Plat-A cells in 60 mm dishes with 4 mL of Dulbecco’s modified Eagle’s medium (DMEM). The next day, when the cells reach 80% - 90% confluence, transfect the cells wit....

Results

A characterization of HT29 cells stably expressing DR3 is shown in Figure 1. The clones express varying levels of DR3, while the wild-type cells, which serve as a negative control, do not show exogenous gene expression. Here we only show five clones, among which clone 1 and 5 express the highest level of DR3. We chose clone 1 and 5 for the following experiments.

The morphology of HT29 and HT29-DR3 c.......

Discussion

In this manuscript, we describe a method to generate HT29-DR3 stable cell lines. HT29-DR3 cells provide a model for studying the molecular mechanisms by which DR3 contributes to apoptosis induced by antimitotic agents. This approach is versatile and repeatable. To ensure the success of the procedure, four key steps of the protocol need to be considered. First, in order to produce a high enough virus titer, it is recommended to perform DNA transfection when the Plat-A cells are at 80% - 90% confluence. Second, the viral s.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
DMEM	Invitrogen	C11965500BT	Supplemented with 10% FBS and 1% Penicilin/Streptomycin
Luminescent Cell Viability Assay	Promega	G7571
Paclitaxel	Selleck	S1150
pMXs-IRES-Blasicidin	Cell Biolabs	RTV-016
Dimethyl sulfoxide	Sigma	D2650
PBS	Invitrogen	C14190500BT
Trypsin-EDTA (0.25%)	Invitrogen	25200056
1,5-Dimethyl-1,5-diazaundecamethylene polymethobromide	Santa Cruz	sc-134220
Transfection Reagent	Promega	E2311
Anti-mouse secondary antibody	Jackson ImmumoResearch	115-035-166
anti-Flag antibody	Sigma	F-3165
HT29 cell line	ATCC	HTB-38
plat-A cell line	Cell Biolabs	RV-102
PVDF Immobilon-P	Millipore	IPVH00010
Cloning Cylinder	Sigma	C1059
Cell Imaging Multi-Mode Reader	Biotek	BTCYT3MV
CKX53 Inverted Microscope	Olympus	CKX53
12-well cell culture plates	Nest	712001
60 mm cell culture plates	Nest	705001
15 mL centrifuge tubes	Nest	601052
0.22 μm steril filter	Millipore	SLGP033RB
Centrifuge 5810 R	Eppendorf	5810000327
96 Well White Polystyrene Microplate	Corning	3903
Western ECL Substrate	BIO-RAD	1705060
ImageQuant LAS 4000	GE Healthcare	ImageQuant LAS 4000
Decoloring Shaker	HINOTECH	TS-2000A
Blasticidin	InvivoGen	ant-bl-1
Automated cell counter	BIO-RAD	TC 20
Opti-MEM I Reduced Serum Medium	Thermo Fisher	31985070