Intravital imaging of fluorescent markers and FRET probes by DNA tattooing

Adriaan D Bins,Jonathan R Halstead,Ton Nm Schumacher,David M Spencer,Jacco Van Rheenen,Kees Jalink,John Bag Haanen,Nullin Divecha

doi:10.1186/1472-6750-7-2

Abstract

BackgroundAdvances in fluorescence microscopy and mouse transgenesis have made it possible to image molecular events in living animals. However, the generation of transgenic mice is a lengthy process and intravital imaging requires specialized knowledge and equipment. Here, we report a rapid and undemanding intravital imaging method using generally available equipment.ResultsBy DNA tattooing we transfect keratinocytes of living mice with DNA encoding fluorescent biosensors. Subsequently, the behavior of individual cells expressing these biosensors can be visualized within hours and using conventional microscopy equipment. Using this "instant transgenic" model in combination with a corrected coordinate system, we followed the in vivo behavior of individual cells expressing either FRET- or location-based biosensors for several days. The utility of this approach was demonstrated by assessment of in vivo caspase-3 activation upon induction of apoptosis.ConclusionThis "instant skin transgenic" model can be used to follow the in vivo behavior of individual cells expressing either FRET- or location-based probes for several days after tattooing and provides a rapid and inexpensive method for intravital imaging in murine skin.

Highlights

Advances in fluorescence microscopy and mouse transgenesis have made it possible to image molecular events in living animals
In an effort to determine the number of cells that express the introduced transgene upon this type of skin application, we tattooed a small area (10 × 15 mm) of the abdominal skin of mice with DNA encoding a GFP reporter protein driven by the CMV immediate early (CMV-IE) promoter. 3 hours thereafter, mice were anesthetized and analyzed for DNA tattoo-induced GFP expression by intravital imaging using a conventional confocal microscope
Expression of the GFP reporter gene was observed in approximately 160 cells per 10 mm2 tattooed skin, showing that the transfection efficiency of DNA tattooing was sufficient for imaging purposes

Summary

Introduction

Advances in fluorescence microscopy and mouse transgenesis have made it possible to image molecular events in living animals. The generation of transgenic mice is a lengthy process and intravital imaging requires specialized knowledge and equipment. We report a rapid and undemanding intravital imaging method using generally available equipment. We present a rapid and inexpensive DNA tattoo method to target FP-genes to keratinocytes in the skin of mice. The behavior of these keratinocytes expressing fluorescent or FRET markers were followed for multiple days with subcellular resolution using a standard confocal microscope. The utility of this tattoo-approach was demonstrated by in vivo imaging of caspase-3 activation upon apoptosis induction

Methods

Results

Conclusion