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Genetic and spectrally distinct in vivo imaging: embryonic stem cells and mice with widespread expression of a monomeric red fluorescent protein.
BMC Biotechnol. 2005 Jul 04; 5:20.BB

Abstract

BACKGROUND

DsRed the red fluorescent protein (RFP) isolated from Discosoma sp. coral holds much promise as a genetically and spectrally distinct alternative to green fluorescent protein (GFP) for application in mice. Widespread use of DsRed has been hampered by several issues resulting in the inability to establish and maintain lines of red fluorescent protein expressing embryonic stem cells and mice. This has been attributed to the non-viability, or toxicity, of the protein, probably as a result of its obligate tetramerization. A mutagenesis approach directing the stepwise evolution of DsRed has produced mRFP1, the first true monomer. mRFP1 currently represents an attractive autofluorescent reporter for use in heterologous systems.

RESULTS

We have used embryonic stem cell-mediated transgenesis to evaluate mRFP1 in embryonic stem cells and mice. We find that mRFP1 exhibits the most spatially homogenous expression when compared to the native (tetrameric) and variant dimeric forms of DsRed. High levels of mRFP1 expression do not affect cell morphology, developmental potential or viability and fertility of animals. High levels of widespread mRFP1 expression are maintained in a constitutive manner in embryonic stem cells in culture and in transgenic animals. We have used various optical imaging modalities to visualize mRFP1 expressing cells in culture, in embryos and adult mice. Moreover co-visualization of red, green and cyan fluorescent cells within a sample is easily achieved without the need for specialized methodologies, such as spectral deconvolution or linear unmixing.

CONCLUSION

Fluorescent proteins with excitation and/or emission profiles in the red part of the visible spectrum represent distinct partners, or longer wavelength substitutes for GFP. Not only do DsRed-based RFPs provide a genetically and spectrally distinct addition to the available repertoire of autoflorescent proteins, but by virtue of their spectral properties they permit deeper tissue imaging. Our work in generating CAG::mRFP1 transgenic ES cells and mice demonstrates the developmental neutrality of mRFP1 in an organismal context. It paves the way for the use of DsRed-based monomeric RFPs in transgenic and gene targeted approaches which often necessitate spatially and/or temporally restricted reporter expression. Moreover animals of the CAG::mRFP1 transgenic strain serve as a source of RFP tagged tissue for the derivation of cell lines and explant, transplant and embryo chimera experiments.

Authors+Show Affiliations

Developmental Biology Program, Sloan-Kettering Institute, New York, NY 10021, USA. jzl2102@columbia.eduNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

15996270

Citation

Long, Jonathan Z., et al. "Genetic and Spectrally Distinct in Vivo Imaging: Embryonic Stem Cells and Mice With Widespread Expression of a Monomeric Red Fluorescent Protein." BMC Biotechnology, vol. 5, 2005, p. 20.
Long JZ, Lackan CS, Hadjantonakis AK. Genetic and spectrally distinct in vivo imaging: embryonic stem cells and mice with widespread expression of a monomeric red fluorescent protein. BMC Biotechnol. 2005;5:20.
Long, J. Z., Lackan, C. S., & Hadjantonakis, A. K. (2005). Genetic and spectrally distinct in vivo imaging: embryonic stem cells and mice with widespread expression of a monomeric red fluorescent protein. BMC Biotechnology, 5, 20.
Long JZ, Lackan CS, Hadjantonakis AK. Genetic and Spectrally Distinct in Vivo Imaging: Embryonic Stem Cells and Mice With Widespread Expression of a Monomeric Red Fluorescent Protein. BMC Biotechnol. 2005 Jul 4;5:20. PubMed PMID: 15996270.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Genetic and spectrally distinct in vivo imaging: embryonic stem cells and mice with widespread expression of a monomeric red fluorescent protein. AU - Long,Jonathan Z, AU - Lackan,Chantal S, AU - Hadjantonakis,Anna-Katerina, Y1 - 2005/07/04/ PY - 2005/05/10/received PY - 2005/07/04/accepted PY - 2005/7/6/pubmed PY - 2006/2/24/medline PY - 2005/7/6/entrez SP - 20 EP - 20 JF - BMC biotechnology JO - BMC Biotechnol. VL - 5 N2 - BACKGROUND: DsRed the red fluorescent protein (RFP) isolated from Discosoma sp. coral holds much promise as a genetically and spectrally distinct alternative to green fluorescent protein (GFP) for application in mice. Widespread use of DsRed has been hampered by several issues resulting in the inability to establish and maintain lines of red fluorescent protein expressing embryonic stem cells and mice. This has been attributed to the non-viability, or toxicity, of the protein, probably as a result of its obligate tetramerization. A mutagenesis approach directing the stepwise evolution of DsRed has produced mRFP1, the first true monomer. mRFP1 currently represents an attractive autofluorescent reporter for use in heterologous systems. RESULTS: We have used embryonic stem cell-mediated transgenesis to evaluate mRFP1 in embryonic stem cells and mice. We find that mRFP1 exhibits the most spatially homogenous expression when compared to the native (tetrameric) and variant dimeric forms of DsRed. High levels of mRFP1 expression do not affect cell morphology, developmental potential or viability and fertility of animals. High levels of widespread mRFP1 expression are maintained in a constitutive manner in embryonic stem cells in culture and in transgenic animals. We have used various optical imaging modalities to visualize mRFP1 expressing cells in culture, in embryos and adult mice. Moreover co-visualization of red, green and cyan fluorescent cells within a sample is easily achieved without the need for specialized methodologies, such as spectral deconvolution or linear unmixing. CONCLUSION: Fluorescent proteins with excitation and/or emission profiles in the red part of the visible spectrum represent distinct partners, or longer wavelength substitutes for GFP. Not only do DsRed-based RFPs provide a genetically and spectrally distinct addition to the available repertoire of autoflorescent proteins, but by virtue of their spectral properties they permit deeper tissue imaging. Our work in generating CAG::mRFP1 transgenic ES cells and mice demonstrates the developmental neutrality of mRFP1 in an organismal context. It paves the way for the use of DsRed-based monomeric RFPs in transgenic and gene targeted approaches which often necessitate spatially and/or temporally restricted reporter expression. Moreover animals of the CAG::mRFP1 transgenic strain serve as a source of RFP tagged tissue for the derivation of cell lines and explant, transplant and embryo chimera experiments. SN - 1472-6750 UR - https://www.unboundmedicine.com/medline/citation/15996270/Genetic_and_spectrally_distinct_in_vivo_imaging:_embryonic_stem_cells_and_mice_with_widespread_expression_of_a_monomeric_red_fluorescent_protein_ L2 - https://bmcbiotechnol.biomedcentral.com/articles/10.1186/1472-6750-5-20 DB - PRIME DP - Unbound Medicine ER -