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Use of the viral 2A peptide for bicistronic expression in transgenic mice.

Trichas G, Begbie J, Srinivas S - BMC Biol. (2008)

Bottom Line: This is currently done by using an internal ribosomal entry site to separate the different coding regions. 2A peptides result in the co-translational 'cleavage' of proteins and are an attractive alternative to the internal ribosomal entry site.Myr-TdTomato and H2B-GFP segregate to mutually exclusive cellular compartments in all tissues examined from a broad range of developmental stages, ranging from embryo to adult.One transgenic line shows X-linked inheritance of the transgene and mosaic expression in females but uniform expression in males, indicating that the transgene has integrated into the X chromosome in this line.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Physiology Anatomy and Genetics, University of Oxford, Oxford, UK. georgios.trichas@dpag.ox.ac.uk

ABSTRACT

Background: Transgenic animals are widely used in biomedical research and biotechnology. Multicistronic constructs, in which several proteins are encoded by a single messenger RNA, are commonly used in genetically engineered animals. This is currently done by using an internal ribosomal entry site to separate the different coding regions. 2A peptides result in the co-translational 'cleavage' of proteins and are an attractive alternative to the internal ribosomal entry site. They are more reliable than the internal ribosomal entry site and lead to expression of multiple cistrons at equimolar levels. They work in a wide variety of eukaryotic cells, but to date have not been demonstrated to function in transgenic mice in an inheritable manner.

Results: To test 2A function in transgenic mice and uncover any possible toxicity of widespread expression of the 2A peptide, we made a bicistronic reporter construct containing the coding sequence for a membrane localised red fluorescent protein (Myr-TdTomato) and a nuclear localised green fluorescent protein (H2B-GFP), separated by a 2A sequence. When this reporter is transfected into HeLa cells, the two fluorescent proteins correctly localise to mutually exclusive cellular compartments, demonstrating that the bicistronic construct is a reliable readout of 2A function. The two fluorescent proteins also correctly localise when the reporter is electroporated into chick neural tube cells. We made two independent transgenic mouse lines that express the bicistronic reporter ubiquitously. For both lines, transgenic mice are born in Mendelian frequencies and are found to be healthy and fertile. Myr-TdTomato and H2B-GFP segregate to mutually exclusive cellular compartments in all tissues examined from a broad range of developmental stages, ranging from embryo to adult. One transgenic line shows X-linked inheritance of the transgene and mosaic expression in females but uniform expression in males, indicating that the transgene has integrated into the X chromosome in this line.

Conclusion: The 2A peptide efficiently mediates co-translational cleavage in transgenic mice in which it has been inherited through the germ-line. Mice expressing it ubiquitously throughout development and into adulthood appear normal. It is therefore a viable tool for use in genetically engineered mice and represents a superior alternative to the widely used internal ribosomal entry site.

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Testing the Tom-2A-GFP construct in chick embryos. (A-C) Three-dimensional volume rendering of a confocal image stack of embryonic chick neural crest cells electroporated with the Tom-2A-GFP construct under the control of a CMV enhancer. The enhanced green fluorescent protein (EGFP) and TdTomato segregate to the nucleus and plasma membrane respectively, indicating that the 2A peptide functions in chick cells. (A) EGFP fluorescence. (B) TdTomato fluorescence. (C) Merge of EGFP (green) and TdTomato (magenta) fluorescence. Scale indicators are rendered in perspective along with the confocal image data and do not translate reliably to a two-dimensional image, therefore, no scale bar is depicted. As an approximate indicator, the two cells at the right of the image that have just divided are, together, between 15 and 20 μm long.
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Figure 2: Testing the Tom-2A-GFP construct in chick embryos. (A-C) Three-dimensional volume rendering of a confocal image stack of embryonic chick neural crest cells electroporated with the Tom-2A-GFP construct under the control of a CMV enhancer. The enhanced green fluorescent protein (EGFP) and TdTomato segregate to the nucleus and plasma membrane respectively, indicating that the 2A peptide functions in chick cells. (A) EGFP fluorescence. (B) TdTomato fluorescence. (C) Merge of EGFP (green) and TdTomato (magenta) fluorescence. Scale indicators are rendered in perspective along with the confocal image data and do not translate reliably to a two-dimensional image, therefore, no scale bar is depicted. As an approximate indicator, the two cells at the right of the image that have just divided are, together, between 15 and 20 μm long.

Mentions: In order to confirm that the Tom-2A-GFP construct worked not only in cultured cells but also in endogenous vertebrate cells, we electroporated it into the neural tube of chick embryos, labelling delaminating neural crest cells. In confirmation of the results with the HeLa cells, EGFP localised to the nucleus and TdTomato to the plasma membrane in electroporated cells, indicating that the 2A peptide functions in chick cells (Figure 2).


Use of the viral 2A peptide for bicistronic expression in transgenic mice.

Trichas G, Begbie J, Srinivas S - BMC Biol. (2008)

Testing the Tom-2A-GFP construct in chick embryos. (A-C) Three-dimensional volume rendering of a confocal image stack of embryonic chick neural crest cells electroporated with the Tom-2A-GFP construct under the control of a CMV enhancer. The enhanced green fluorescent protein (EGFP) and TdTomato segregate to the nucleus and plasma membrane respectively, indicating that the 2A peptide functions in chick cells. (A) EGFP fluorescence. (B) TdTomato fluorescence. (C) Merge of EGFP (green) and TdTomato (magenta) fluorescence. Scale indicators are rendered in perspective along with the confocal image data and do not translate reliably to a two-dimensional image, therefore, no scale bar is depicted. As an approximate indicator, the two cells at the right of the image that have just divided are, together, between 15 and 20 μm long.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2553761&req=5

Figure 2: Testing the Tom-2A-GFP construct in chick embryos. (A-C) Three-dimensional volume rendering of a confocal image stack of embryonic chick neural crest cells electroporated with the Tom-2A-GFP construct under the control of a CMV enhancer. The enhanced green fluorescent protein (EGFP) and TdTomato segregate to the nucleus and plasma membrane respectively, indicating that the 2A peptide functions in chick cells. (A) EGFP fluorescence. (B) TdTomato fluorescence. (C) Merge of EGFP (green) and TdTomato (magenta) fluorescence. Scale indicators are rendered in perspective along with the confocal image data and do not translate reliably to a two-dimensional image, therefore, no scale bar is depicted. As an approximate indicator, the two cells at the right of the image that have just divided are, together, between 15 and 20 μm long.
Mentions: In order to confirm that the Tom-2A-GFP construct worked not only in cultured cells but also in endogenous vertebrate cells, we electroporated it into the neural tube of chick embryos, labelling delaminating neural crest cells. In confirmation of the results with the HeLa cells, EGFP localised to the nucleus and TdTomato to the plasma membrane in electroporated cells, indicating that the 2A peptide functions in chick cells (Figure 2).

Bottom Line: This is currently done by using an internal ribosomal entry site to separate the different coding regions. 2A peptides result in the co-translational 'cleavage' of proteins and are an attractive alternative to the internal ribosomal entry site.Myr-TdTomato and H2B-GFP segregate to mutually exclusive cellular compartments in all tissues examined from a broad range of developmental stages, ranging from embryo to adult.One transgenic line shows X-linked inheritance of the transgene and mosaic expression in females but uniform expression in males, indicating that the transgene has integrated into the X chromosome in this line.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Physiology Anatomy and Genetics, University of Oxford, Oxford, UK. georgios.trichas@dpag.ox.ac.uk

ABSTRACT

Background: Transgenic animals are widely used in biomedical research and biotechnology. Multicistronic constructs, in which several proteins are encoded by a single messenger RNA, are commonly used in genetically engineered animals. This is currently done by using an internal ribosomal entry site to separate the different coding regions. 2A peptides result in the co-translational 'cleavage' of proteins and are an attractive alternative to the internal ribosomal entry site. They are more reliable than the internal ribosomal entry site and lead to expression of multiple cistrons at equimolar levels. They work in a wide variety of eukaryotic cells, but to date have not been demonstrated to function in transgenic mice in an inheritable manner.

Results: To test 2A function in transgenic mice and uncover any possible toxicity of widespread expression of the 2A peptide, we made a bicistronic reporter construct containing the coding sequence for a membrane localised red fluorescent protein (Myr-TdTomato) and a nuclear localised green fluorescent protein (H2B-GFP), separated by a 2A sequence. When this reporter is transfected into HeLa cells, the two fluorescent proteins correctly localise to mutually exclusive cellular compartments, demonstrating that the bicistronic construct is a reliable readout of 2A function. The two fluorescent proteins also correctly localise when the reporter is electroporated into chick neural tube cells. We made two independent transgenic mouse lines that express the bicistronic reporter ubiquitously. For both lines, transgenic mice are born in Mendelian frequencies and are found to be healthy and fertile. Myr-TdTomato and H2B-GFP segregate to mutually exclusive cellular compartments in all tissues examined from a broad range of developmental stages, ranging from embryo to adult. One transgenic line shows X-linked inheritance of the transgene and mosaic expression in females but uniform expression in males, indicating that the transgene has integrated into the X chromosome in this line.

Conclusion: The 2A peptide efficiently mediates co-translational cleavage in transgenic mice in which it has been inherited through the germ-line. Mice expressing it ubiquitously throughout development and into adulthood appear normal. It is therefore a viable tool for use in genetically engineered mice and represents a superior alternative to the widely used internal ribosomal entry site.

Show MeSH
Related in: MedlinePlus