We have investigated the expression and germline transmission of native mRFP1 in embryonic stem cells and mice. This is an essential prerequisite to using the available suite of DsRed-derived monomeric RFPs in fusions as reporters for high-resolution in vivo imaging, and in particular, as fusion proteins exhibiting subcellular localization and acting as segmental markers of 3-dimensional space.
As a first step we cloned the mRFP1
gene into a vector permitting widespread expression in a variety of cells types in culture and in vivo
in mice. The mRFP1
coding sequence was engineered to contain a Kozak consensus sequence at its 5' end and subsequently introduced into pCAGGS
a vector utilizing the chicken beta actin promoter and SV40
immediate early enhancer combination, designed to drive high-level constitutive gene expression in ES cells, embryos and adult mice [17
]. Standard protocols were used to establish stable CAG::mRFP1
transgenic lines of ES cells constitutively expressing mRFP1.
Fluorescent colonies (CAG::mRFP1
transgenic) were identified and picked under an epifluorescence stereo dissecting microscope. Clones were passaged in 96-well plates, and scored for the maintenance and extent of red fluorescence with maintenance in culture. Clones that failed to meet these criteria were discarded from further analysis. Thereafter only clones exhibiting robust transgene expression in vitro
under both stem cell and differentiation conditions were further analyzed for level and heterogeneity of red fluorescence by flow cytometry as described previously for GFP-variants [19
]. Only those clones exhibiting high levels of homogenous expression were selected for further analysis. Sustained strong homogenous red fluorescence and retention of normal ES cell morphology when grown on either gelatin-coated plates or mouse embryo fibroblast feeders was a prerequisite feature of these cells
We then co-cultured three transgenic ES cell lines expressing different fluorescent proteins, namely; CAG::ECFP
along with wild type (non-fluorescent) ES cells, and then visualized fluorescence in resulting chimeric colonies (Fig. ). The different fluorophores could easily be distinguished both with standard filter sets using epifluorescence optics and confocal microscopy, without the need for special image processing such as spectral deconvolution or linear unmixing [20
]. We also noted that comparable levels of fluorescence were produced as all three color variants produced fluorescent signal within the same dynamic range.
Figure 1 Co-visualization of multiple fluorescent proteins in mouse embryonic stem (ES) cells. Mixed colony of embryonic stem (ES) cells comprised of wild type (untagged) cells, CAG::ECFP transgenic cells exhibiting widespread expression of ECFP, CAG::EGFP transgenic (more ...)
Having established the neutrality of widespread mRFP1 expression in ES cells, we went on to use ES cell mediated transgenesis through the generation of germline transmitting chimeras to introduce the CAG::mRFP1
transgene into mice. As a first step to test the extent of expression of the transgene in embryos, we generated 4n (tetraploid) wild type <-> mRFP1 ES cell derived chimeras [21
], exactly as described previously [3
]. Resulting, completely ES cell-derived embryos exhibited widespread mRFP1 expression, indicating that the level of expression was sufficiently strong to be visualized, that the transgene was not silenced and that development was able to proceed normally to midgestation (data not shown). To produce germline transmitting chimeric adult animals, we next generated diploid wild type <-> mRFP1 ES cell chimeras that were allowed to go to term. The CAG::mRFP1
transgene was transmitted to F1 offspring in a Mendelian fashion, suggesting that widespread mRFP1 expressing is compatible with normal development and fertility. Two ES cell lines were taken germline and shown to produce an equivalent intensity and range of fluorescence, therefore data from only one line are shown.
Wide field epifluorescent and laser scanning confocal microscopy was used to image this constitutively expressed transgene reporter in preimplantation stage mouse embryos hemizygous (Tg/+) for the CAG::mRFP1 transgene (Fig. and additional files). Such non-invasive visualization in living preparations allowed us to acquire high-magnification, sequential optical sections (z-stacks) that were used to generate high-resolution anatomical, volumetric images of embryos. To do this, stacks of sequential optical sections were computationally reconstructed into 3-dimensional (3D) projections. This methodology was used to generate 3D image sets, and is illustrated here by imaging whole mouse embryos at the 1-cell stage and blastocyst stage. These data sets can be computationally manipulated in various ways including for the visualization of individual xy slices from a z-stack (Fig. and and additional files), or of rendered images from the full (Fig. ), or partial z-stack (Fig. and ). It should be noted that even using epifluorescence imaging CAG::mRFP1 embryos were clearly distinguished from stage-matched CAG::EGFP embryos (Fig. ).
Figure 2 RFP expression in CAG::mRFP1 preimplantation stage embryos. Single CAG::mRFP1 Tg/+ zygote including the second polar body (A–E). A single x-y section taken from a z-stack, bright field image (A), overlay single confocal section of red fluorescence (more ...)
Wide field microscopic imaging of later stage hemizygous embryos illustrated the robust, homogenous and widespread expression of mRFP1 from early postimplantation, embryonic day (E) 6.5 (Fig. ) to later fetal stages in both the embryo proper and extraembryonic lineages, including the placenta (Fig. ). Dissection of organs from fetuses confirmed widespread red fluorescence in CAG::mRFP1/+ organs contrasted with a lack of signal observed in non-transgenic littermates (Fig. and ).
Figure 3 RFP expression in CAG::mRFP1 postimplantation embryos. Bright field and dark field epifluorescent images of CAG::mRFP1 Tg/+ embryos and non-transgenic littermates at E6.5 (A), E7.75 (B), E8.75 (C). CAG::mRFP1 Tg/+ embryos, CAG::EGFP Tg/+ embryos and non-transgenic (more ...)
Examination of newborn animals revealed strong widespread expression of mRFP1 in the skin of hemizygous CAG::mRFP/+ transgenics and demonstrated that this red fluorophore can be distinguished from green fluorescence observed in CAG::EGFP/+ transgenics, and from non-transgenic littermates by standard macroscopic visualization using standard filter sets and epiflourescent excitation (Fig. ).
Figure 4 Transgenic CAG::mRFP1 mice can be distinguished from CAG::EGFP animals. Macroscopic images of non-transgenic, CAG::mRFP1 Tg/+ and CAG::EGFP Tg/+ newborn (P5) mouse pups demonstrating that red fluorescence can clearly be distinguished from green fluorescence (more ...)
Further analysis of various adult organs, including the peritoneum, heart, lung, eye, brain, liver, pancreas, spleen and kidney, that were freshly obtained from CAG::mRFP1/+
adult animals revealed robust and widespread fluorescence (Fig. ), as has been reported for animals expressing GFP-based fluorescent proteins under the regulation of the CAG promoter. We also noted that newborn pups, or bald skin and tissues expressing mRFP1, exhibit a pink coloration under normal light when compared to non-transgenic or CAG::EGFP
]. This is particularly evident in albino animals (tails in Fig. ), and in unpigmented organs such as the brain and pancreas (Fig. and ). We believe this results from mRFP1 being a red fluorphore with a spectrum closer to the visible range, so that it can be visualized as a pink pigmentation under daylight or bright field conditions. Consequently, newborn CAG::mRFP1
pups can be genotyped based on their pink pigmentation, alleviating the need to image them under epifluorescent conditions.
Figure 5 Widespread RFP expression in adult organs. Panels of bright field and corresponding dark field epifluorescent images of organs taken from a 4 week old CAG::mRFP1 Tg/+ mouse and a non-transgenic littermate. Peritoneum (A), heart (B), lung (C), eye (D), (more ...)
Moreover we succeeded in breeding the CAG::mRFP1 transgene to homozygosity. Homozygous animals retained developmental potential and therefore colonies of the strain are routinely maintained in the homozygous state. To date, we have observed no noticeable reduction in fluorescence or fertility for over four generations on both ICR outbred and 129 inbred backgrounds. From routine daily observations no behavioral differences are distinguished between CAG::mRFP1 animals and wild type age matched animals, though no behavioral tests have been implemented.