Reporters of Gene Expression: Autofluorescent Proteins

Abstract

This unit on GFP provides clear, detailed, easy‐to‐follow directions for successful transduction of GFP into appropriate cell lines, as well as carefully detailed strategy and trouble‐shooting sections. Instructions for sorting and purification of sucessfully transduced cells and for analysis and re‐analysis of cells together with sample data are all included. The discussion provides data on multiple GFP mutants and lists relevant sources and suggested reading material.

Characterization of stably transfected fusion protein GFP‐estrogen receptor‐α in MCF‐7 human breast cancer cells

“…For harvesting, cells were either lyzed on tissue culture plates for protein and RNA analyses or trypsinized and collected for fluorescent-activated cell sorting [Chadee et al, 1995], nuclei preparation, or nuclear matrix preparation [Samuel et al, 1997]. Bivariant analysis of GFP-ER and cell cycle was done as described [Rasko, 2001].…”

Generation of HIV‐1‐Based Lentiviral Vector Particles

“…HT1080 cell line (ATCC #CCL-121) 293T cell growth medium (see recipe) Vector preparations (Basic Protocols 3 or 4 or Alternate Protocols 1 or 2) 6 mg/ml (1000×) polybrene stock solution G418 (UNIT 9.5) 0.3% (w/v) crystal violet in 70% (v/v) methanol 6-well tissue culture plates Additional reagents and equipment for flow cytometric analysis of EGFP expression (Rasko, 1999), Xgal staining for lacZ (β-galactosidase) expression (UNIT 9.10), or enumeration of neo-resistant cells (UNIT 9.5)…”

“…4a. For vectors containing EGFP or laZ marker: After 48 hr, determine the relative end-point vector titers (in TU/ml) by flow cytometric analysis for EGFP expression (if the vector contains the EGFP gene; see, e.g., Rasko, 1999) or by Xgal staining (if the vector contains the lacZ gene; see UNIT 9.10). Use the following equation:…”

“…Growth medium for CEM-GFP cells: IMDM containing 10% FBS (see recipe for serum-containing growth media for transduction) 6 mg/ml polybrene stock solution HIV packaging plasmid DNA (e.g., pCMV∆R8.91) Additional reagents and equipment for flow cytometric analysis of GFP expression (Rasko, 1999) 1. Prepare lentiviral supernatant (see Basic Protocol 6, step 1).…”