Elsevier

Experimental Cell Research

Volume 313, Issue 9, 15 May 2007, Pages 1943-1950
Experimental Cell Research

Research Article
Sensitive detection of GFP utilizing tyramide signal amplification to overcome gene silencing

https://doi.org/10.1016/j.yexcr.2007.02.024Get rights and content

Abstract

The green fluorescent protein (GFP) is among the most commonly used expression markers in biology. GFP-tagged cells have played a particularly important role in studies of cell lineage. Sensitive detection of GFP is crucially important for such studies to be successful, and problems with detection may account for discrepancies in the literature regarding the possible fate choices of stem cells. Here we describe a very sensitive technique for visualization of GFP. Using it we can detect about 90% of cells of donor origin while we could only see about 50% of these cells when we employ the methods that are in general use in other laboratories. In addition, we provide evidence that some cells permanently silence GFP expression. In the case of the progeny of bone marrow stem cells, it appears that the more distantly related they are to their precursors, the more likely it is that they will turn off the lineage marker.

Introduction

The green fluorescent protein (GFP) was discovered as a by product of isolating aequorin from jelly fish by Shimomura et al. [2] in 1962. The importance of the discovery was not obvious until much later; GFP proved to be an excellent protein marker molecule for gene expression (see [2]). Gradually, immunohistochemical (IHC) detection techniques have become more and more sensitive. We can measure and visualize proteins in amounts that were unimaginable 10 years ago. Numerous studies utilized GFP to track cell fate following bone marrow transplantation, local injection or promoter specific expression [3], [4], [5], [6], [7], [8], [9], [10]. While a variety of groups showed that GFP-expressing bone marrow cells are able to seed many tissues and differentiate into tissue specific cells, an equal number of papers failed to confirm those results and stated the opposite [11], [12]. One of the factors that seem to affect chimerism is the presence or absence of tissue injury/disease. In normal, healthy tissue circulating bone marrow cells do not seem to contribute to regeneration as much as when the tissue is in “need” [4]. Furthermore, it was noted by several studies that the expression of GFP is variable; in many instances the expression weakens with time or in some cases GFP becomes undetectable [13]. The possibility that the GFP transgene can be silenced has also been raised [14], [15], [16], [17], [18], [19], [20]. The field has been plagued by controversy mostly due to differences in techniques used by the different groups to follow cell fate as summarized in [21]. In the last decade a new, very sensitive technique became available utilizing tyramide signal amplification [22], [23] and its application to immunohistochemistry was reported [1] describing dilutions of primary antibodies for optimal immunohistochemistry [24] as well as its use in dual immunostaining techniques [25]. Since we also noticed very faint green fluorescent cells in our experimental samples, we decided to apply this technique to attempt to visualize most of the GFP expressing cells. The use of this newly designed, sensitive method might help to clarify the confusion in the literature.

Section snippets

Animal experiments

Female C57B mice were irradiated using 900 rad in two equal doses (irradiation time was 4 min 15 s each time) 8 h apart. Following the second irradiation, the animals were transplanted with bone marrow from male Z/EG (lacZ/EGFP) double reporter transgenic mice [26] that had previously been crossed with a Cre-actin mouse to result in an animal which ubiquitously and stably express the green fluorescent protein. Donor mice were euthanized by decapitation under anesthesia and the bodies were

Results

A one-step immunostaining protocol (using the primary antibody and a fluorochrome-conjugated secondary antibody) for GFP cells (Fig. 2B) yielded higher signal intensity and a crisper image than was seen by imaging native fluorescence (Fig. 2A). The difference between the two images, however, was more qualitative than quantitative. Due to the increase in staining intensity with IHC (depositing more fluorochrome at the antigen site), more cells were readily detectable even with low magnification.

Discussion

The first transgenic mouse was produced by Gordon in 1980 [32]. By constructing transgenes that contained GFP cDNA driven by specific promoter sequences the target proteins could now be identified by detecting the green fluorescence [33]. The use of green fluorescence protein revolutionized the monitoring of gene expression. Many techniques were used and compared to optimize the immunohistochemical detection of GFP using conventional [34] and confocal microscope [35] even in paraffin embedded

Conclusion

Our experiments demonstrate that on the average we fail to detect half of the cells that express GFP if we use only traditional immunostaining. It is interesting to note that the cells that are still strongly fluorescent are microglia—which are known to be of bone marrow origin. We suggest that the loss of GFP may be a function of differentiation, i.e., the less cells resemble their bone marrow precursors, the less GFP is expressed. Based on our results, the maximal GFP sensitivity can be

Acknowledgments

This research was supported by the DIR, NIDCR, NINDS and NIMH of the Intramural Research Program, NIH. Zs.E.T. is also supported by OTKA T 043169. The authors want to acknowledge the help of Joanne Severe with the statistics.

References (41)

  • O. Shimomura

    The discovery of aequorin and green fluorescent protein

    J. Microsc.

    (2005)
  • G.F. Beilhack et al.

    Purified allogeneic hematopoietic stem cell transplantation blocks diabetes pathogenesis in NOD mice

    Diabetes

    (2003)
  • T. Ito et al.

    Application of bone marrow-derived stem cells in experimental nephrology

    Exp. Nephrol.

    (2001)
  • E. Mezey et al.

    Turning blood into brain: cells bearing neuronal antigens generated in vivo from bone marrow

    Science

    (2000)
  • K. Nakano et al.

    Differentiation of transplanted bone marrow cells in the adult mouse brain

    Transplantation

    (2001)
  • C.P. Hofstetter et al.

    Marrow stromal cells form guiding strands in the injured spinal cord and promote recovery

    Proc. Natl. Acad. Sci. U. S. A.

    (2002)
  • K.A. Moore et al.

    Stem cells and their niches

    Science

    (2006)
  • K.A. Jackson et al.

    Stem cells: a minireview

    J. Cell Biochem. Suppl.

    (2002)
  • R.A. McTaggart et al.

    An uncomfortable silence em leader while we all search for a better reporter gene in adult stem cell biology

    Hepatology

    (2004)
  • H. Xia et al.

    siRNA-mediated gene silencing in vitro and in vivo

    Nat. Biotechnol.

    (2002)
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