Transduction to Label PDX Tumor Cells: Introducing Lentivirus Expressing Fluorescent Marker into the Tumor Cell In Vitro

Abstract

Source: Hanna, C., et al. Labeling of Breast Cancer Patient-derived Xenografts with Traceable Reporters for Tumor Growth and Metastasis Studies. J. Vis. Exp. (2016).

The following video describes a technique of transduction to label (patient-derived xenografts) PDX tumor cells, which is  used for introducing lentivirus expressing green-fluorescent protein and luciferase reporters into the tumor cell in vitro.

Protocol

1. Transduction of PDX-derived Tumor Cells Centrifuge dissociated tumor cells at 300 g x 5 min and resuspend in 2 ml mammosphere media. Count viable cells using trypan blue exclusion (Resuspend digested cells in 5 ml wash buffer and count both viable and non-viable cells using trypan blue exclusion. Briefly, mix 50 µl of cells and 50 µl of trypan blue and count trypan-blue excluding cells using a hemocytometer). Prepare 10 ml of mammosphere media containing 8 µg/ml polybrene (2 µ…

Representative Results

Figure 1: Tracking Transduction Efficiency in Dissociated PDX Cells. A) PDX-dissociated cells enriched for human epithelial cells (Lin+ depletion) 24 hr after transduction with pSIH1-H1-copGFP-T2A-puro lentiviral particles. B) PDX-dissociated cells from a separate experiment, without epithelial cell enrichment, 72 hr after transduction with pHAGE-EF1aL-luciferase-…

Materials

DMEM/F12 (1:1)	Hyclone	SH30023.01
bFGF	BD Biosciences	354060
EGF	BD Biosciences	354001
Heparin	Sigma	H4784
B27	Gibco/Thermo Fisher	17504-44
Anti-fungi-antibiotics	Hyclone	SV30010
HBSS Red Ca2+/Mg2+ free	Hyclone	SH30031.02
Hepes
Cultrex	Cultrex	3433-005-01	Basement Matrix Extract (BME)
30 °C shaker	NewBrunswick Scientific CO. INC	Series 25 Incubator Shaker