Many transgenic lines exist that express Cre recombinase either broadly or in cell type or tissue-specific manners (http://nagy.mshri.on.ca/cre/
) making the system widely applicable for inducing cell type- or tissue-specific gene deletions as well as lineage tracing during development. When used for lineage tracing, Cre-expressing lines are crossed to reporter lines in which Cre-mediated excision activates a reporter gene. The efficacy and specificity of Cre excision in gene ablation studies is also frequently monitored with crosses to Cre reporter lines.
Several reporter lines have been developed (Kawamoto et al., 2000
; Lobe et al., 1999
; Luche et al., 2007
; Mao et al., 1999
; Muzumdar et al., 2007
; Novak et al., 2000
; Soriano, 1999
; Srinivas et al., 2001
; Vintersten et al., 2004
; Yamauchi et al., 1999
; Zinyk et al., 1998
). A number of single reporters have, for example, targeted the ROSA26 locus with lacZ (Mao et al., 1999
; Soriano, 1999
) or a variety of fluorescent proteins (Luche et al., 2007
; Mao et al., 2001
; Srinivas et al., 2001
). Single reporters however suffer from the limitation that there is no reporter expression until Cre excision and thus no internal control for expression in cell types or tissues where reporter expression may be weak or the “ubiquitous” promoter may in fact not be active. As an alternative, double reporter lines have been developed in which one reporter is ubiquitously expressed prior to Cre-mediated recombination while a second is activated following Cre excision (Lobe et al., 1999
; Muzumdar et al., 2007
; Novak et al., 2000
; Vintersten et al., 2004
). These mice offer the advantage that one reporter must always be “on” in any individual cell, thus creating an internal control.
Most dual reporter systems described to date have combined an enzymatic reporter (lacZ or alkaline phosphatase) with either green or red fluorescent proteins (Lobe et al., 1999
; Novak et al., 2000
; Vintersten et al., 2004
). Although histochemical or immunocytochemical detection of enzymes such as β-galactosidase is simple, the sensitivity of this method may be problematic and vary depending on the amount of protein synthesized or other factors that can affect its subcellular distribution leading to an underestimation of the extent of staining, particularly in the nervous system (Friedrich et al., 1993
). By contrast, fluorescent proteins offer the advantage that they can be visualized in tissues without fixation and cells can be isolated using fluorescence-activated cell sorting (FACS) without exogenous substrates. However, to date, only one dual reporter using fluorescent proteins has been developed (Muzumdar et al., 2007
). These authors targeted a red fluorescent protein (RFP) from the family of reef coral fluorescent proteins known as tandem dimer Tomato along with an enhanced green fluorescent protein (EGFP) to the ROSA26 locus.
We developed a two-color fluorescent protein reporter by modifying the plasmid used to generate Z/EG mice (Novak et al., 2000
). This vector utilizes the chicken β-actin promoter (CCAG) and first intron coupled to the CMV immediate early enhancer with a downstream polyadenylation signal derived from the rabbit β-globin gene (Niwa et al., 1991
). Downstream of the CCAG promoter is a loxP
flanked lacZ cDNA coupled to an EGFP reporter. We replaced the lacZ cDNA with a cDNA for DsRed-Express, an RFP derived from the Dicosoma sp
. of reef coral fluorescent proteins. These proteins offer the advantage that due to their low homology with the Aequorea
fluorescent proteins (Matz et al., 1999
), specific monoclonal and polyclonal antibodies against them exist that do not cross-react with EGFP. Thus, in addition to spontaneous fluorescence, they can be distinguished from Aequorea
GFPs by immunohistochemical means. To minimize integration site effects and possible promoter interference, we flanked the construct at each end with two paired copies of the 1.2-kb HS4 insulator sequence, found near the 5′-end of the chicken β-globin locus (Chung et al., 1993
; Hebbes et al., 1994
). This insulator has been previously shown to largely protect integrated transgenes from position site effects (Ciana et al., 2001
; Guglielmi et al., 2003
; Potts et al., 2000
). A diagram of the final construct, which we termed IRG, for “insulator/red/green” is shown in .
FIG. 1 RFP expression in transgenic IRG mice. Panel a shows the design of the IRG transgene with insulator sequences (Ins), the chicken-CMV β-actin promoter (CCAG), as well as the DsRed express and EGFP cDNAs indicated. loxP sites are indicated as open (more ...)
Transgenic mice were generated by pronuclear injection of linearized IRG plasmid DNA and analysis of mice from a line established from founder five is described. By semiquantitative PCR, this line was estimated to contain seven copies of the transgene (). Multiple generations of IRG mice from Line 5 have not exhibited any signs of toxicity due to RFP expression up to 15 months of age and both males as well as females are fertile.
Transgenic IRG mice demonstrated red fluorescence under the appropriate fluorescent excitation filter for RFP without any green fluorescence (Figs. and ). All internal organs examined showed red fluorescence without detectable green fluorescence above that seen in nontransgenic embryos. Examples of tissues from both embryonic and adult IRG mice are shown in . Western blotting of tissues from adult IRG mice showed that although the level of DsRed-Express protein varied from tissue to tissue, expression was detectable in all organs examined ().
FIG. 2 Cre-mediated recombination in IRG reporter mice. In (a, b), an E14.5 single transgenic IRG embryo is shown viewed under red (a) or green (b) filters. In (d, e), an IRG/EIIa-Cre double transgenic embryo is shown viewed under red (d) or green (e) filters. (more ...)
To determine the extent of Cre-mediated recombination that could be induced, we crossed IRG mice with either EIIa-Cre (Lakso et al., 1996
) or CMV-Cre mice (Schwenk et al., 1995
), two general Cre deletor lines. EIIa-Cre mice express Cre under the control of the adenovirus EIIa promoter, which drives Cre expression as early as the preimplantation embryo (Lakso et al., 1996
), whereas in CMV-Cre mice, Cre expression is driven by the human cytomegalovirus minimal promoter (Schwenk et al., 1995
). Both lines have been shown to be efficient inducers of widespread Cre-mediated recombination. As shown in , the embryo as well as the placenta from double transgenic IRG/EIIa-Cre mice showed green fluorescence. Widespread EGFP activation was also seen in IRG/CMV-Cre mice (Supplementary Fig. S1
). Therefore widespread Cre-mediated activation of EGFP is possible in IRG mice, thus demonstrating their suitability as a general Cre reporter.
We next examined Cre-mediated excision in the central nervous system by crossing IRG mice to Nestin-Cre mice. Nestin is expressed in neural stem/progenitor cells and regarded as a general marker of this cell population. Nestin expression in the nervous system is controlled by an intron 2 enhancer that has been widely used to express heterologous genes in the neural stem/progenitor cell population (Zimmerman et al., 1994
). In double transgenic IRG/Nestin-Cre embryos, EGFP expression was activated in brain and spinal cord (). Internal organs exhibited only spotty green fluorescence in heart and kidneys (data not shown). Double transgenic mice showed EGFP activation throughout the brain (). The only grossly nonrecombined region was an area of the dorsal midbrain (), which based on immunohistochemical staining corresponds to prominent RFP expression within the superior colliculus (Supplementary Fig. S2
Immunohistochemical staining of single transgenic IRG mice showed widespread RFP expression throughout the adult brain without any EGFP expression. Examples of RFP staining in the hippocampus and cerebellum of IRG single transgenic mice are shown in . In double transgenic IRG/Nestin-Cre mice, immunohistochemical staining documented widespread EGFP activation in both embryonic and adult brain as expected for a Nestin-Cre transgene. Although EGFP activation did not occur in all cells, the pattern of activation was very similar to that observed in Nestin-Cre crosses to Z/EG reporter mice (see ). IRG mice should thus be suitable for two-color fluorescent protein imaging in the brain as well as other organs.
FIG. 3 DsRed expression in adult brain of single transgenic IRG mice. Shown is immunohistochemical staining for DsRed in CA1 pyramidal neurons of the hippocampus (a), Purkinje cells of the cerebellum (b), and the hippocampal granule cell layer (c). Scale bar: (more ...)
FIG. 4 EGFP activation in the adult brain of IRG/Nestin-Cre double transgenic mice. Pyramidal cells in the CA1 region of the hippocampus from Nestin-Cre mice crossed to ether IRG (a–c) or Z/EG (d–f) reporter mice. In (a–c) sections were (more ...)
Having two fluorescent proteins offers the opportunity of using FACS to separate recombined from nonrecombined cell populations without the need for exogenous substrates. To determine whether RFP and EGFP expressing cells could be separated using IRG mice, we collected E14.5 embryonic brain from IRG as well as IRG crossed to Nestin-Cre mice. The tissues were dissociated into single cell suspensions and analyzed by FACS using cells from nontransgenic littermates to set levels of background fluorescence (). Using a 488-nm excitation laser, 54.6% of the cells sorted from the IRG brains were RFP positive. We suspect that the relatively low percentage of RFP positive cells reflects the fact that optimal excitation for DsRed-Express is 557 nm (Biosciences, 2003
) and thus using the available 488 nm laser, we likely failed to detect weakly positive cells. Supporting this notion when we immunostained single cell suspensions from brains of the same-age embryos, we found that 96% of the cells were DsRed-immunolabeled (). Muzumdar et al. (2007)
have reported a similar discrepancy between detection of tandem dimer tomato positive cells by FACS and visual inspection when using a 488-nm laser for FACS detection. No EGFP positive cells were detected either by FACS and or by immunostaining in single transgenic IRG mice.
FIG. 5 Analysis of single cell suspensions from embryonic brain of IRG and IRG/Nestin-Cre transgenic mice by immunostaining and FACS. In (a) the telencephalon from brains of E14.5 IRG single transgenic or IRG crossed to Nestin-Cre double transgenic mice were (more ...)
When Nestin-Cre crossed to IRG, mice were examined by FACS (), the number of EGFP positive cells was very similar between FACS (79%) and visual inspection of immunostained single cell suspensions (84%) likely reflecting that the optimal excitation for EGFP is 484 nm (Biosciences, 2003
), which is much closer to that of the laser available for FACS detection. In these crosses, 0.8% of cells were RFP positive (i.e., nonrecombined) by FACS analysis and 14% RFP positive by immunostaining (see ), again likely representing an under estimation of RFP expressing cells by FACS.
Structural changes in the brain occur in normal aging and with a variety of pathological states. Increasingly it is being realized that detailed reconstructions of brain structure at various levels of resolution can contribute to understanding disease states. Such imaging requires imaging in thick materials, whole brains, or thick tissue slabs. Immunolabeling within thick sections is often compromised because of the poor tissue penetration of most immunostaining methods. Fluorescent proteins offer the possibility of overcoming the limitations of immunostaining by allowing native fluorescence to be imaged in situ. In addition, having two fluorescent proteins in the IRG mouse and the ability to activate them selectively based on the choice of Cre driver creates the possibility of imaging distinct structures or cell types within the same section. Nestin-Cre, for example, activates widespread EGFP expression in neurons but because the nestin intron 2 enhancer is not active in vascular elements (Wen et al., 2005
), cerebral blood vessels which are mesenchymally derived remain nonrecombined and red. Thus, neurons and vascular structures can in principle be imaged simultaneously.
Two of us (PRH and SLW) recently described algorithms for reconstructing 3D images from original grayscale data obtained by laser-scanning confocal microscopy (Rodriguez et al., 2006
; Wearne et al., 2005
). Using double transgenic IRG/Nestin-Cre mice, we determined whether these algorithms could be used to image native fluorescence in thick sections of brain tissue to visualize simultaneously neuronal and vascular structures. Using 200-μm thick sections of brain tissue, native RFP and EGFP fluorescence was imaged by laser-scanning confocal microscopy (see Materials and Methods). shows high-resolution confocal imaging of the CA1 pyramidal layer of the hippocampus. Recombined pyramidal neurons appear green and nonrecombined blood vessels are red. An additional example of simultaneous vascular and neuronal imaging in the cerebellar cortex is shown in Supplementary Figure S3
FIG. 6 Maximal two-dimensional projection of a three-dimensional reconstruction of deconvolved and merged z-stacks of native red and green fluorescence from 200-μm thick tissue sections using confocal laser-scanning microscopy. The CA1 pyramidal cell (more ...)
Native fluorescence offers the advantage that it can be detected in thick tissue sections at depths where immunostaining is difficult. To determine whether native expression and immunostaining have comparable sensitivities at tissue thicknesses where immunostaining is possible, we took sections of brain from EIIa-Cre mice crossed to IRG mice where recombination is essentially complete and performed immunostaining for EGFP using an AlexaFluor568
-conjugated secondary. We then imaged native EGFP fluorescence (green) and EGFP immunostaining (red) in the same sections. The patterns of EGFP expression detected by native fluorescence and immunostaining appeared essentially identical. An example of imaging from the hippocampal granule cell layer is shown in Supplementary Figure S4
. To address whether RFP imaging had similar sensitivities, sections of brain from single transgenic IRG mice were immuno-stained for DsRed-Express using an AlexaFluor488
-conjugated secondary antibody and then native RFP fluorescence (red) and DsRed-Express immunostaining (green) were compared. The patterns of RFP expression detected by native fluorescence and immunostaining were essentially identical (Supplementary Fig. 4
). Thus detection of EGFP and RFP by native fluorescence and immunostaining appear equally sensitive under the conditions tested.
These studies show that in addition to serving as a general reporter for Cre-mediated recombination, IRG mice can be used to image complex cellular relationships in thick tissue sections, which cannot currently be reliably achieved by standard immunostaining approaches. By choice of Cre driver it should be possible to image a variety of complex cellular relationships in situ. IRG mice should thus provide a versatile tool for a variety of applications utilizing the Cre/loxP recombination system. Future modifications of this system could also be envisioned whereby modulation of the temporal onset of recombination could be regulated to study processes such as adult neurogenesis.
Finally it is of note that to our knowledge this is the first reporter line created using pronuclear injection as opposed to targeting an endogenous gene locus by homologous recombination success that we suspect may be attributed to the inclusion of the insulator sequences. If so, insulator sequences may provide a mechanism for expressing other reporter constructs without the need for gene targeting.