Embryonic stem cells and mice expressing different GFP variants for multiple non-invasive reporter usage within a single animal
© Hadjantonakis et al; licensee BioMed Central Ltd. 2002
Received: 28 April 2002
Accepted: 11 June 2002
Published: 11 June 2002
Non-invasive autofluorescent reporters have revolutionized lineage labeling in an array of different organisms. In recent years green fluorescent protein (GFP) from the bioluminescent jellyfish Aequoria Victoria has gained popularity in mouse transgenic and gene targeting regimes . It offers several advantages over conventional gene-based reporters, such as lacZ and alkaline phosphatase, in that its visualization does not require a chromogenic substrate and can be realized in vivo. We have previously demonstrated the utility and developmental neutrality of enhanced green fluorescent protein (EGFP) in embryonic stem (ES) cells and mice .
In this study we have used embryonic stem (ES) cell-mediated transgenesis to test the enhanced cyan fluorescent protein (ECFP) and enhanced yellow fluorescent protein (EYFP), two mutant and spectrally distinct color variants of wild type (wt) GFP. We have also tested DsRed1, the novel red fluorescent protein reporter recently cloned from the Discostoma coral by virtue of its homology to GFP. To this end, we have established lines of ES cells together with viable and fertile mice having widespread expression of either the ECFP or EYFP GFP-variant reporters. However, we were unable to generate equivalent DsRed1 lines, suggesting that DsRed1 is not developmentally neutral or that transgene expression cannot be sustained constitutively. Balanced (diploid <-> diploid) and polarized (tetraploid <-> diploid) chimeras comprising combinations of the ECFP and EYFP ES cells and/or embryos, demonstrate that populations of cells expressing each individual reporter can be distinguished within a single animal.
GFP variant reporters are unique in allowing non-invasive multi-spectral visualization in live samples. The ECFP and EYFP-expressing transgenic ES cells and mice that we have generated provide sources of cells and tissues for combinatorial, double-tagged recombination experiments, chimeras or transplantations.
The ease of manipulating its genome has helped establish the mouse as the premier model organism for mammalian genetic studies. Both directed and random mutagenesis approaches, including the technologies of transgenesis and gene targeting in ES cells, have become commonplace. Genetically manipulated mice, often incorporating gene-based reporters, are frequently being used to model and understand mammalian development and disease processes. Fluorescent protein reporters currently represent a superior alternative to other gene-based reporters such as the bacterial lacZ or human placental alkaline phosphatase in that their visualization is non-invasive, and as such does not require chromogenic substrates. Fluorescence can be monitored in real-time in vivo and in situ, and has the added advantage in that it can be quantified. The prototype fluorescent protein reporter is green fluorescent protein (GFP, reviewed in ), which is derived from the bioluminescent jellyfish Aequorea Victoria .
Green fluorescent variants of wild type GFP (wtGFP) with improved thermostability and fluorescence emission, including enhanced green fluorescent protein (EGFP, ), and mMGFP , have gained popularity for use in mice. Recently however, several additional mutants of wtGFP, with altered excitation and emission spectral profiles (i.e. fluoresce in colors other than green), as well as improved thermostability and fluorescence, have been described [4, 6].
Dual color imaging of several GFP variant proteins is a very attractive prospect and has become a standard procedure in cell biology [7, 8]. It has also recently been demonstrated in a subset of cells within the central nervous system (CNS) of mice using both transgenic and viral infection-based approaches [9, 10]. We therefore wished to investigate the feasibility of non-invasive multiple reporter imaging in mice, and as a first step we wanted to ascertain whether we could establish viable and fertile mice with widespread expression of GFP variant reporters.
Results and discussion
We tested several of the available wtGFP variants for developmental neutrality and stability of widespread expression. An ES cell-mediated transgenic approach was used to evaluate the Enhanced Cyan Fluorescent Protein (ECFP) and Enhanced Yellow Fluorescent Protein (EYFP) wtGFP spectral variants [4, 11]. ECFP harbors six amino substitutions (Y66W, F64L, S65T, N146I, M153T, V163A) as compared to GFP, whereas EYFP harbors four (S65G, V68L, S72A and T203Y). In addition to increasing the quantum yield and solubility of the proteins these mutations shift the spectral profiles of the translation products [4, 11].
A breakthrough in the availability of spectrally distinct autofluorescent proteins came with the cloning of six anthozoan fluorescent proteins all having 20–30% identity to wtGFP . Of these, DsRed1 (referred to as drFP585 by Matz et al., ), a 28 kDa protein isolated from the IndoPacific sea anemone Discosoma sp, posseses the longest excitation (558 nm) and emission (583 nm) wavelength maxima known for a wild-type autofluorescent protein. The sequence of DsRed1 has recently been modified and optimized for high levels of expression in mammalian cells by the introduction of 144 silent base pair changes corresponding to human codon usage preferences. We therefore also tested this novel red fluorescent protein (RFP) in ES cells and mice.
Zygotic expression of the FP transgenes is clearly evident by the blastocyst stage (Fig. 2a,2b,2c,2d) and remains widespread thereafter (Fig. 2). Figure 2 demonstrates that ECFP+ embryos can easily be distinguished from EYFP+ embryos and from non-transgenic non-fluorescent embryos from preimplantation stages (Fig. 2a,2b,2c,2d) to postimplantation stages (Fig. 2e,2f,2g,2h). Additionally adult mice can also be discriminated on the basis of the color of their fluorescence (Fig. 2i,2j,2k).
Even though our RFP transgenic ES cell lines failed to maintain strong uniform expression with in vitro passage, we selected three lines exhibiting the most robust expression for making chimeras, in order to ascertain whether we could achieve, or possibly recover, RFP expression in animals. Cells were flow sorted prior to aggregation so as to enrich for RFP expression. Even though they had lost their characteristic ES cell morphology all three RFP+ ES cell lines went gemline as detected by coat color and PCR for the DsRed1 transgene (data not shown). However expression of the RFP reporter could not be detected in any organ of adult mice nor in embryos (data not shown). This suggests that the DsRed1 RFP reporter was unable to recapitulate the expression profile of the promoter/enhancer combination employed. Therefore it appears that GFP variant reporters are more versatile with respect to their application in mice.
From these observations we conclude that the blue-shifted ECFP, and the red-shifted EYFP variants of wtGFP are both amenable to use in mice. Also since we have established lines of viable and fertile mice having widespread FP expression we have demonstrated that ECFP and EYFP represent developmentally neutral reporters. Since we were unable to obtain ES cells, embryos or mice with widespread expression of a RFP (DsRed1) we conclude that this reporter is not developmentally neutral or that its expression cannot be sustained at levels high enough for detection, and as such is not as versatile as other gene-based fluorescent reporters.
ECFP and EYFP can be distinguished on the basis of their unique non-overlapping spectral profiles, and chimeras comprised of ECFP and EYFP tagged compartments demonstrate that the two reporters can be simultaneously non-invasively visualized within the same embryo or adult organ. Therefore it should be possible to use flow cytometry to isolate cells expressing either or both of the reporters from embryos or adult organs .
These two reporters can also be used to create pairs of donors and acceptors for in vivo fluorescence energy transfer (FRET) . Since the emission profiles of ECFP and EYFP are the least overlapping of all the usable GFP variants, these ECFP and EYFP currently represent the best combination for co-visualization and for use in multi-spectral double-tagged transgenic, chimeric and/or in vivo FRET experiments (reviewed in ).
These experiments have demonstrated the feasibility of monitoring multiple FP reporters non-invasively within a single animal, and have shown the developmental neutrality of the FP reporters employed. This should pave the way for novel strategies designed to incorporate the use of compound transgenics or tissue recombination or chimeric analyses where several compartments are to be tagged. This technology should provide new possibilities in manipulating the mouse genome, and non-invasively studying the consequences.
Materials and methods
cDNAs encoding ECFP, EYFP and DsRed were amplified from plasmids pECFP, pEYFP and pDsRed1-N1 (Clontech, Inc.) by PCR using HiFi Taq Polymerase (Invitrogen). For ECFP and EYFP primers used were GFP-L (5'-TTG AAT TCG CCA CCA TGG TGA GC) and GFP-R (5'-TTG AAT TCT TAC TTG TAC AGC TCG TCC), for dsRed primers were red-L (5'-TTG AAT TCG CCA CCA TGG TGC GCT C) and red-R (5'-TTG AAT TCT TAC GCT ACA GCT ACA GGA ACA GGT G). All 5' primers contain a Kozak consensus translation initiation site upstream of the ATG for increased translation efficiency in eukaryotic cells . To construct vectors driving ubiquitous transgene expression in ES cells and mice, the PCR amplified cDNAs were digested with EcoRI and inserted into the EcoRI sites of pCAGGS  to generate pCX-ECFP, pCX-EYFP and pCX-DsRed. pCAGGS contains the chicken beta-actin promoter and first intron coupled to the CMV immediate early enhancer, and rabbit beta-globin polyadenylation signal. This promoter/enhancer combination has previously been shown to drive strong and widespread transgene expression in ES cells, embryos and adult mice [18, 19].
R1 ES cells  were maintained under standard culture conditions in the presence of LIF, as described previously . 20 μg of SalI linearized pCX-ECFP, pCX-EYFP or pCX-DsRed1 was co-electroporated into 2 × 107 cells with 5–10 μg of circular pPGKPuro . This provided transient drug resistance which could be used as a selection for obtaining individual colonies. Selection was applied 24 hours after electroporation and maintained for 5 days, thereafter cells were propagated in in the absence of selection. Fluorescent colonies were picked into 96-well plates, expanded and archived as described previously (. Clones were screened for strong, stable, and homogenous transgene expression during culture in the absence of drug selection.
Chimera production and germline transmission
ES cell lines exhibiting robust transgene expression in vitro were assessed in vivo for the spatiotemporal extent of transgene expression and their developmental potential in chimeras produced by aggregation with wild type tetraploid ICR embryos so as to produce completely ES cell-derived embryos, as described previously [20, 23]. A detailed protocol can be found on the world wide web at http://www.mshri.on.ca/nagy/. Clones giving fluorescent E12.5 embryos having no readily identifiable malformations were used to generate germline transmitting chimeras by aggregation with diploid wild type ICR embryos as described previously [21, 23]. After germline transmission transgenes were bred to homozygosity. Transgenes were maintained on both mixedbred ICR and inbred 129/SvJ genetic backgrounds .
Photographs of live samples, freshly dissected in PBS ( were taken on a Leica MZFLIII stereo dissecting microscope equipped with epifluorescence optics. Filter sets used were: excitation 436/20 nm, dichroic mirror 455 nm, barrier 480/40 nm for ECFP, excitation 500/20 nm, dichroic mirror 515 nm, barrier 535/30 nm for EYFP and excitation 540/25 nm, dichroic mirror 565 nm, barrier 605/55 for DsRed. Adult mice were anaesthetized with avertin. They were then placed on the stage of a stereo dissecting microscope (Leica MZFLIII) from which the objective had been removed, such that they were illuminated using the epifluorescence optics (lamp and excitation filters). Photographs were taken using a Nikon FE10 35 mm SLR camera fitted with individual emission filters. Images were processed using Photoshop software (Adobe Systems).
The cyan and yellow fluorescent protein-expressing mice described here have been acquired by, and are available from, the Jackson Laboratory Induced Mutant Resource http://www.jax.org/resources/documents/imr/. They are referred to therein as 003373 Stock TgN(ActbECFP) 1Nagy (CK6/ECFP) and 003772 Stock TgN(ActbEYFP)1Nagy (YC5/EYFP).
We thank J.-I. Miyazaki for the pCAGGS plasmid. A.-K.H. is particularly indebted to V. E. Papaioannou for helpful discussion and constructive comments on the manuscript. This work was supported by grants from the National Cancer Institute of Canada.
- Chalfie M, Tu Y, Euskirchen G, Ward WW, Prasher DC: Green fluorescent protein as a marker for gene expression. Science. 1994, 263: 802-5.View ArticleGoogle Scholar
- Hadjantonakis AK, Gertsenstein M, Ikawa M, Okabe M, Nagy A: Generating green fluorescent mice by germline transmission of green fluorescent ES cells. Mech Dev. 1998, 76: 79-90. 10.1016/S0925-4773(98)00093-8.View ArticleGoogle Scholar
- Chalfie M: Green fluorescent protein. Photochem Photobiol. 1995, 62: 651-6.View ArticleGoogle Scholar
- Cormack BP, Valdivia RH, Falkow S: FACS-optimized mutants of the green fluorescent protein (GFP). Gene. 1996, 173: 33-8. 10.1016/0378-1119(95)00685-0.View ArticleGoogle Scholar
- Zernicka-Goetz M, Pines J, McLean Hunter S, Dixon JP, Siemering KR, Haseloff J, Evans MJ: Following cell fate in the living mouse embryo. Development. 1997, 124: 1133-7.Google Scholar
- Yang TT, Cheng L, Kain SR: Optimized codon usage and chromophore mutations provide enhanced sensitivity with the green fluorescent protein. Nucleic Acids Res. 1996, 24: 4592-3. 10.1093/nar/24.22.4592.View ArticleGoogle Scholar
- Yang TT, Sinai P, Green G, Kitts PA, Chen YT, Lybarger L, Chervenak R, Patterson GH, Piston DW, Kain SR: Improved fluorescence and dual color detection with enhanced blue and green variants of the green fluorescent protein. J Biol Chem. 1998, 273: 8212-6. 10.1074/jbc.273.14.8212.View ArticleGoogle Scholar
- Ellenberg J, Lippincott-Schwartz J, Presley JF: Dual-colour imaging with GFP variants. Trends Cell Biol. 1999, 9: 52-6. 10.1016/S0962-8924(98)01420-2.View ArticleGoogle Scholar
- Feng G, Mellor RH, Bernstein M, Keller-Peck C, Nguyen QT, Wallace M, Nerbonne JM, Lichtman JW, Sanes JR: Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP. Neuron. 2000, 28: 41-51. 10.1016/S0896-6273(00)00084-2.View ArticleGoogle Scholar
- Furuta T, Tomioka R, Taki K, Nakamura K, Tamamaki N, Kaneko T: In Vivo Transduction of Central Neurons Using Recombinant Sindbis Virus. Golgi-like labeling of dendrites and axons with membrane-targeted fluorescent proteins. J Histochem Cytochem. 2001, 49: 1497-508.View ArticleGoogle Scholar
- Heim R, Tsien RY: Engineering green fluorescent protein for improved brightness, longer wavelengths and fluorescence resonance energy transfer. Curr Biol. 1996, 6: 178-82. 10.1016/S0960-9822(02)00450-5.View ArticleGoogle Scholar
- Matz MV, Fradkov AF, Labas YA, Savitsky AP, Zaraisky AG, Markelov ML, Lukyanov SA: Fluorescent proteins from nonbioluminescent Anthozoa species. Nat Biotechnol. 1999, 17: 969-73. 10.1038/13657.View ArticleGoogle Scholar
- Wood SA, Allen ND, Rossant J, Auerbach A, Nagy A: Non-injection methods for the production of embryonic stem cell-embryo chimaeras. Nature. 1993, 365: 87-9. 10.1038/365087a0.View ArticleGoogle Scholar
- Hadjantonakis AK, Nagy A: FACS for the isolation of individual cells from transgenic mice harboring a fluorescent protein reporter. Genesis. 2000, 27: 95-8. 10.1002/1526-968X(200007)27:3<95::AID-GENE10>3.0.CO;2-A.View ArticleGoogle Scholar
- Pollok BA, Heim R: Using GFP in FRET-based applications. Trends Cell Biol. 1999, 9: 57-60. 10.1016/S0962-8924(98)01434-2.View ArticleGoogle Scholar
- Hadjantonakis AK, Nagy A: The color of mice: in the light of GFP-variant reporters. Histochem Cell Biol. 2001, 115: 49-58.View ArticleGoogle Scholar
- Kozak M: Nucleotide sequences of 5'-terminal ribosome-protected initiation regions from two reovirus messages. Nature. 1977, 269: 391-4.View ArticleGoogle Scholar
- Niwa H, Yamamura K, Miyazaki J: Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene. 1991, 108: 193-9. 10.1016/0378-1119(91)90434-D.View ArticleGoogle Scholar
- Okabe M, Ikawa M, Kominami K, Nakanishi T, Nishimune Y: 'Green mice' as a source of ubiquitous green cells. FEBS Lett. 1997, 407: 313-9. 10.1016/S0014-5793(97)00313-X.View ArticleGoogle Scholar
- Nagy A, Rossant J, Nagy R, Abramow-Newerly W, Roder JC: Derivation of completely cell culture-derived mice from early-passage embryonic stem cells. Proc Natl Acad Sci U S A. 1993, 90: 8424-8.View ArticleGoogle Scholar
- Pirity M, Hadjantonakis AK, Nagy A: Embryonic stem cells, creating transgenic animals. Methods Cell Biol. 1998, 57: 279-93.View ArticleGoogle Scholar
- Tucker KL, Beard C, Dausmann J, Jackson-Grusby L, Laird PW, Lei H, Li E, Jaenisch R: Germ-line passage is required for establishment of methylation and expression patterns of imprinted but not of nonimprinted genes. Genes Dev. 1996, 10: 1008-20.View ArticleGoogle Scholar
- Tanaka M, Hadjantonakis AK, Nagy A: Aggregation chimeras. Combining ES cells, diploid and tetraploid embryos. Methods Mol Biol. 2001, 158: 135-54. 10.1385/1-59259-220-1:135.Google Scholar
- Hogan BLM, Beddington RSP, Costantini FL: Manipulating the mouse embryo. A laboratory manual. Cold Spring Harbor, NY.: Cold Spring Harbor Laboratory Press.;. 1994Google Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL.