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A BAC-bacterial recombination method to generate physically linked multiple gene reporter DNA constructs.

Maye P, Stover ML, Liu Y, Rowe DW, Gong S, Lichtler AC - BMC Biotechnol. (2009)

Bottom Line: Moreover, gene linkage allows for their common chromosomal integration into a single locus.However, the testing of this multi-reporter DNA construct by transgenesis does suggest that the linkage of two different genes together, despite their large size, can still create a positional effect.We believe that gene choice, genomic DNA fragment size and the presence of endogenous insulator elements are critical variables.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Reconstructive Sciences, Center for Regenerative Medicine, University of Connecticut Health Center, Farmington, CT, USA. pmaye@neuron.uchc.edu

ABSTRACT

Background: Reporter gene mice are valuable animal models for biological research providing a gene expression readout that can contribute to cellular characterization within the context of a developmental process. With the advancement of bacterial recombination techniques to engineer reporter gene constructs from BAC genomic clones and the generation of optically distinguishable fluorescent protein reporter genes, there is an unprecedented capability to engineer more informative transgenic reporter mouse models relative to what has been traditionally available.

Results: We demonstrate here our first effort on the development of a three stage bacterial recombination strategy to physically link multiple genes together with their respective fluorescent protein (FP) reporters in one DNA fragment. This strategy uses bacterial recombination techniques to: (1) subclone genes of interest into BAC linking vectors, (2) insert desired reporter genes into respective genes and (3) link different gene-reporters together. As proof of concept, we have generated a single DNA fragment containing the genes Trap, Dmp1, and Ibsp driving the expression of ECFP, mCherry, and Topaz FP reporter genes, respectively. Using this DNA construct, we have successfully generated transgenic reporter mice that retain two to three gene readouts.

Conclusion: The three stage methodology to link multiple genes with their respective fluorescent protein reporter works with reasonable efficiency. Moreover, gene linkage allows for their common chromosomal integration into a single locus. However, the testing of this multi-reporter DNA construct by transgenesis does suggest that the linkage of two different genes together, despite their large size, can still create a positional effect. We believe that gene choice, genomic DNA fragment size and the presence of endogenous insulator elements are critical variables.

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Functional characterization of linked multi-reporter gene construct in transgenic mice. (A) Imaging of fluorescent reporters in six week old spine under fixed exposure demonstrating intensity differences among different lines. Ibsp-Topaz and Dmp1-mCherry fluorescent reporter genes are easily detected in most lines, however Trap-ECFP in most lines appears highly mosaic expression with only a random cell having ECFP fluorescence. Line 28-2-2 is the only line that has uniform Trap-ECFP reporter expression, but it has no reporter expression for Dmp1 and low expression for Ibsp-Topaz (over exposed inset). (B&C) Confirming the fidelity of the Trap-ECFP reporter. (B) TRAP histological stain on a 3 week old femur that was initially imaged (C) for Trap-ECFP expression. ECFP expression and TRAP staining are essentially identical. (D-E) Genotyping of litters from three separate mouse lines for all three reporter genes. Individual reporters co-segregate together verifying common chromosomal integration. For lines 26-1-5 and 28-4-5 all three reporters are detected, however, in line 28-2-2 DMP1-mCherry reporter is not detected by PCR (D) or viewing of tail biopsies (E). (F) Estimated copy number of DNA construct in different transgenic lines using primers directed against the TRAP reporter. (G) High magnification expression in Ibsp-Topaz and Dmp1-mCherry in trabecular bone of a 6 week old female femur from line 28-4-3.
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Figure 4: Functional characterization of linked multi-reporter gene construct in transgenic mice. (A) Imaging of fluorescent reporters in six week old spine under fixed exposure demonstrating intensity differences among different lines. Ibsp-Topaz and Dmp1-mCherry fluorescent reporter genes are easily detected in most lines, however Trap-ECFP in most lines appears highly mosaic expression with only a random cell having ECFP fluorescence. Line 28-2-2 is the only line that has uniform Trap-ECFP reporter expression, but it has no reporter expression for Dmp1 and low expression for Ibsp-Topaz (over exposed inset). (B&C) Confirming the fidelity of the Trap-ECFP reporter. (B) TRAP histological stain on a 3 week old femur that was initially imaged (C) for Trap-ECFP expression. ECFP expression and TRAP staining are essentially identical. (D-E) Genotyping of litters from three separate mouse lines for all three reporter genes. Individual reporters co-segregate together verifying common chromosomal integration. For lines 26-1-5 and 28-4-5 all three reporters are detected, however, in line 28-2-2 DMP1-mCherry reporter is not detected by PCR (D) or viewing of tail biopsies (E). (F) Estimated copy number of DNA construct in different transgenic lines using primers directed against the TRAP reporter. (G) High magnification expression in Ibsp-Topaz and Dmp1-mCherry in trabecular bone of a 6 week old female femur from line 28-4-3.

Mentions: To determine the functionality of the linked three gene reporter construct, transgenic mice were generated by DNA pronuclear injection. Nine of thirty potential founder mice showed expression of one or more of the three reporters by examining the tail biopsy under a standard epifluorescence microscope. Three transgenic founders expressed all reporter genes at a very low level and therefore were euthanized. F2 generation animals from the six remaining transgenic founder lines are currently being characterized and future work will report on their properties in greater detail. However, our preliminary observations of these transgenic lines suggest the outcome of this technical approach to design multiple reporter gene constructs has met with mixed success. Imaging of 6 week old female spines across multiple lines under the same exposure conditions within each reporter demonstrates that F2 animals from lines 26-1-5, 28-4-3, 28-4-5, 28-2-4, 28-2-5 express Ibsp-Topaz and Dmp1-mCherry relatively strong while showing lower to undetectable levels of Trap-ECFP expression (Fig. 4A). Occasionally, a Trap-ECFP positive cell can be observed suggesting mosaic expression of the Trap reporter (Fig. 4A, Trap-ECFP). In contrast, line 28-2-2 expresses Trap-ECFP at higher levels and uniformly in osteoclasts as determined by comparing histological TRAP staining to Trap-ECFP (compare Fig. 4B to 4C), while showing low expression of Ibsp and no detectable expression of Dmp1 (Fig. 4A, inset images).


A BAC-bacterial recombination method to generate physically linked multiple gene reporter DNA constructs.

Maye P, Stover ML, Liu Y, Rowe DW, Gong S, Lichtler AC - BMC Biotechnol. (2009)

Functional characterization of linked multi-reporter gene construct in transgenic mice. (A) Imaging of fluorescent reporters in six week old spine under fixed exposure demonstrating intensity differences among different lines. Ibsp-Topaz and Dmp1-mCherry fluorescent reporter genes are easily detected in most lines, however Trap-ECFP in most lines appears highly mosaic expression with only a random cell having ECFP fluorescence. Line 28-2-2 is the only line that has uniform Trap-ECFP reporter expression, but it has no reporter expression for Dmp1 and low expression for Ibsp-Topaz (over exposed inset). (B&C) Confirming the fidelity of the Trap-ECFP reporter. (B) TRAP histological stain on a 3 week old femur that was initially imaged (C) for Trap-ECFP expression. ECFP expression and TRAP staining are essentially identical. (D-E) Genotyping of litters from three separate mouse lines for all three reporter genes. Individual reporters co-segregate together verifying common chromosomal integration. For lines 26-1-5 and 28-4-5 all three reporters are detected, however, in line 28-2-2 DMP1-mCherry reporter is not detected by PCR (D) or viewing of tail biopsies (E). (F) Estimated copy number of DNA construct in different transgenic lines using primers directed against the TRAP reporter. (G) High magnification expression in Ibsp-Topaz and Dmp1-mCherry in trabecular bone of a 6 week old female femur from line 28-4-3.
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Figure 4: Functional characterization of linked multi-reporter gene construct in transgenic mice. (A) Imaging of fluorescent reporters in six week old spine under fixed exposure demonstrating intensity differences among different lines. Ibsp-Topaz and Dmp1-mCherry fluorescent reporter genes are easily detected in most lines, however Trap-ECFP in most lines appears highly mosaic expression with only a random cell having ECFP fluorescence. Line 28-2-2 is the only line that has uniform Trap-ECFP reporter expression, but it has no reporter expression for Dmp1 and low expression for Ibsp-Topaz (over exposed inset). (B&C) Confirming the fidelity of the Trap-ECFP reporter. (B) TRAP histological stain on a 3 week old femur that was initially imaged (C) for Trap-ECFP expression. ECFP expression and TRAP staining are essentially identical. (D-E) Genotyping of litters from three separate mouse lines for all three reporter genes. Individual reporters co-segregate together verifying common chromosomal integration. For lines 26-1-5 and 28-4-5 all three reporters are detected, however, in line 28-2-2 DMP1-mCherry reporter is not detected by PCR (D) or viewing of tail biopsies (E). (F) Estimated copy number of DNA construct in different transgenic lines using primers directed against the TRAP reporter. (G) High magnification expression in Ibsp-Topaz and Dmp1-mCherry in trabecular bone of a 6 week old female femur from line 28-4-3.
Mentions: To determine the functionality of the linked three gene reporter construct, transgenic mice were generated by DNA pronuclear injection. Nine of thirty potential founder mice showed expression of one or more of the three reporters by examining the tail biopsy under a standard epifluorescence microscope. Three transgenic founders expressed all reporter genes at a very low level and therefore were euthanized. F2 generation animals from the six remaining transgenic founder lines are currently being characterized and future work will report on their properties in greater detail. However, our preliminary observations of these transgenic lines suggest the outcome of this technical approach to design multiple reporter gene constructs has met with mixed success. Imaging of 6 week old female spines across multiple lines under the same exposure conditions within each reporter demonstrates that F2 animals from lines 26-1-5, 28-4-3, 28-4-5, 28-2-4, 28-2-5 express Ibsp-Topaz and Dmp1-mCherry relatively strong while showing lower to undetectable levels of Trap-ECFP expression (Fig. 4A). Occasionally, a Trap-ECFP positive cell can be observed suggesting mosaic expression of the Trap reporter (Fig. 4A, Trap-ECFP). In contrast, line 28-2-2 expresses Trap-ECFP at higher levels and uniformly in osteoclasts as determined by comparing histological TRAP staining to Trap-ECFP (compare Fig. 4B to 4C), while showing low expression of Ibsp and no detectable expression of Dmp1 (Fig. 4A, inset images).

Bottom Line: Moreover, gene linkage allows for their common chromosomal integration into a single locus.However, the testing of this multi-reporter DNA construct by transgenesis does suggest that the linkage of two different genes together, despite their large size, can still create a positional effect.We believe that gene choice, genomic DNA fragment size and the presence of endogenous insulator elements are critical variables.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Reconstructive Sciences, Center for Regenerative Medicine, University of Connecticut Health Center, Farmington, CT, USA. pmaye@neuron.uchc.edu

ABSTRACT

Background: Reporter gene mice are valuable animal models for biological research providing a gene expression readout that can contribute to cellular characterization within the context of a developmental process. With the advancement of bacterial recombination techniques to engineer reporter gene constructs from BAC genomic clones and the generation of optically distinguishable fluorescent protein reporter genes, there is an unprecedented capability to engineer more informative transgenic reporter mouse models relative to what has been traditionally available.

Results: We demonstrate here our first effort on the development of a three stage bacterial recombination strategy to physically link multiple genes together with their respective fluorescent protein (FP) reporters in one DNA fragment. This strategy uses bacterial recombination techniques to: (1) subclone genes of interest into BAC linking vectors, (2) insert desired reporter genes into respective genes and (3) link different gene-reporters together. As proof of concept, we have generated a single DNA fragment containing the genes Trap, Dmp1, and Ibsp driving the expression of ECFP, mCherry, and Topaz FP reporter genes, respectively. Using this DNA construct, we have successfully generated transgenic reporter mice that retain two to three gene readouts.

Conclusion: The three stage methodology to link multiple genes with their respective fluorescent protein reporter works with reasonable efficiency. Moreover, gene linkage allows for their common chromosomal integration into a single locus. However, the testing of this multi-reporter DNA construct by transgenesis does suggest that the linkage of two different genes together, despite their large size, can still create a positional effect. We believe that gene choice, genomic DNA fragment size and the presence of endogenous insulator elements are critical variables.

Show MeSH
Related in: MedlinePlus