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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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Step 2: Insertion of fluorescent protein reporter genes. (A) Diagrams showing the shuttle vectors containing different spectral FP variants for the specified genes and the two step recombination methodology. Homology arms for Ibsp (Arms A & B), Dmp1 (Arms C & D) and Trap (Arms E & F) were cloned into pLD53-Topaz, pLD53-mCherry, and pLD53-ECFP. Recombination was selected for using ampicillin plus gentamicin or spectinomycin. Sucrose resolution was carried out to remove unwanted vector sequences including ampicillin resistance. (B) Diagram of ECFP insertion into TRAP. (C) ECFP insertion introduced a unique Not1 site verifying integration into GM-Trap. (D) Diagram of Topaz insertion into Ibsp followed by mCherry insertion into Dmp1. (E) Cla1 restriction digestion of Dmp1/Ibsp subcloned fragment at progressive stages of reporter gene insertion. The insertion of mCherry introduces an additional Cla1 site. (F) Diagram of diagnostic PCR carried out with primers flanking the homology arms and fluorescent protein coding regions for all three genes. (G) PCR amplification comparing original BAC clones to fluorescent protein reporter BAC clones. PCR product sizes without reporter gene insertion were 1426 bp for Dmp1, 1569 bp for Ibsp, and 1450 bp for Trap. PCR product sizes with reporter gene insertion were 2559 bp for Dmp1, 2299 bp for Ibsp, and 2545 bp for Trap.
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Figure 2: Step 2: Insertion of fluorescent protein reporter genes. (A) Diagrams showing the shuttle vectors containing different spectral FP variants for the specified genes and the two step recombination methodology. Homology arms for Ibsp (Arms A & B), Dmp1 (Arms C & D) and Trap (Arms E & F) were cloned into pLD53-Topaz, pLD53-mCherry, and pLD53-ECFP. Recombination was selected for using ampicillin plus gentamicin or spectinomycin. Sucrose resolution was carried out to remove unwanted vector sequences including ampicillin resistance. (B) Diagram of ECFP insertion into TRAP. (C) ECFP insertion introduced a unique Not1 site verifying integration into GM-Trap. (D) Diagram of Topaz insertion into Ibsp followed by mCherry insertion into Dmp1. (E) Cla1 restriction digestion of Dmp1/Ibsp subcloned fragment at progressive stages of reporter gene insertion. The insertion of mCherry introduces an additional Cla1 site. (F) Diagram of diagnostic PCR carried out with primers flanking the homology arms and fluorescent protein coding regions for all three genes. (G) PCR amplification comparing original BAC clones to fluorescent protein reporter BAC clones. PCR product sizes without reporter gene insertion were 1426 bp for Dmp1, 1569 bp for Ibsp, and 1450 bp for Trap. PCR product sizes with reporter gene insertion were 2559 bp for Dmp1, 2299 bp for Ibsp, and 2545 bp for Trap.

Mentions: Different bacterial recombination strategies exist to insert a reporter gene into a gene of interest. We favored using a two step recombination strategy developed by Heintz and co-workers (Fig. 2A) because it involves a resolution step to remove unwanted vector sequences and antibiotic selection genes allowing us to repeatedly target multiple genes within the same genomic DNA fragment [8]. The shuttle vector (diagramed in Fig. 2A) has positive (ampicillin) and negative (Sac B) selection schemes that allows for insertion of the vector and resolution of unwanted vector sequences in two consecutive recombination steps (Fig. 2A). We modified the original pLD53-SCAEB vector by changing the original EGFP with the spectrally distinct FP reporters, Topaz, mCherry, and ECFP (Fig. 2A). To avoid the creation of undesireable regions of homology, we also decided to engineer our FP reporters with three different polyadenylation sequences, SV40, bovine growth hormone, and β-globin for ECFP, Topaz, and mCherry, respectively. Furthermore, we decided to keep Topaz and ECFP, Aequorea victoria GFP variants, which have nearly identical DNA sequences, separate by recombining them into the two different BAC subclones. Recombination between Topaz and ECFP during linkage (Fig. 3) is less of concern since this step involves the use of the Red recombinase system, which works by a gap end repair mechanism.


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)

Step 2: Insertion of fluorescent protein reporter genes. (A) Diagrams showing the shuttle vectors containing different spectral FP variants for the specified genes and the two step recombination methodology. Homology arms for Ibsp (Arms A & B), Dmp1 (Arms C & D) and Trap (Arms E & F) were cloned into pLD53-Topaz, pLD53-mCherry, and pLD53-ECFP. Recombination was selected for using ampicillin plus gentamicin or spectinomycin. Sucrose resolution was carried out to remove unwanted vector sequences including ampicillin resistance. (B) Diagram of ECFP insertion into TRAP. (C) ECFP insertion introduced a unique Not1 site verifying integration into GM-Trap. (D) Diagram of Topaz insertion into Ibsp followed by mCherry insertion into Dmp1. (E) Cla1 restriction digestion of Dmp1/Ibsp subcloned fragment at progressive stages of reporter gene insertion. The insertion of mCherry introduces an additional Cla1 site. (F) Diagram of diagnostic PCR carried out with primers flanking the homology arms and fluorescent protein coding regions for all three genes. (G) PCR amplification comparing original BAC clones to fluorescent protein reporter BAC clones. PCR product sizes without reporter gene insertion were 1426 bp for Dmp1, 1569 bp for Ibsp, and 1450 bp for Trap. PCR product sizes with reporter gene insertion were 2559 bp for Dmp1, 2299 bp for Ibsp, and 2545 bp for Trap.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 2: Step 2: Insertion of fluorescent protein reporter genes. (A) Diagrams showing the shuttle vectors containing different spectral FP variants for the specified genes and the two step recombination methodology. Homology arms for Ibsp (Arms A & B), Dmp1 (Arms C & D) and Trap (Arms E & F) were cloned into pLD53-Topaz, pLD53-mCherry, and pLD53-ECFP. Recombination was selected for using ampicillin plus gentamicin or spectinomycin. Sucrose resolution was carried out to remove unwanted vector sequences including ampicillin resistance. (B) Diagram of ECFP insertion into TRAP. (C) ECFP insertion introduced a unique Not1 site verifying integration into GM-Trap. (D) Diagram of Topaz insertion into Ibsp followed by mCherry insertion into Dmp1. (E) Cla1 restriction digestion of Dmp1/Ibsp subcloned fragment at progressive stages of reporter gene insertion. The insertion of mCherry introduces an additional Cla1 site. (F) Diagram of diagnostic PCR carried out with primers flanking the homology arms and fluorescent protein coding regions for all three genes. (G) PCR amplification comparing original BAC clones to fluorescent protein reporter BAC clones. PCR product sizes without reporter gene insertion were 1426 bp for Dmp1, 1569 bp for Ibsp, and 1450 bp for Trap. PCR product sizes with reporter gene insertion were 2559 bp for Dmp1, 2299 bp for Ibsp, and 2545 bp for Trap.
Mentions: Different bacterial recombination strategies exist to insert a reporter gene into a gene of interest. We favored using a two step recombination strategy developed by Heintz and co-workers (Fig. 2A) because it involves a resolution step to remove unwanted vector sequences and antibiotic selection genes allowing us to repeatedly target multiple genes within the same genomic DNA fragment [8]. The shuttle vector (diagramed in Fig. 2A) has positive (ampicillin) and negative (Sac B) selection schemes that allows for insertion of the vector and resolution of unwanted vector sequences in two consecutive recombination steps (Fig. 2A). We modified the original pLD53-SCAEB vector by changing the original EGFP with the spectrally distinct FP reporters, Topaz, mCherry, and ECFP (Fig. 2A). To avoid the creation of undesireable regions of homology, we also decided to engineer our FP reporters with three different polyadenylation sequences, SV40, bovine growth hormone, and β-globin for ECFP, Topaz, and mCherry, respectively. Furthermore, we decided to keep Topaz and ECFP, Aequorea victoria GFP variants, which have nearly identical DNA sequences, separate by recombining them into the two different BAC subclones. Recombination between Topaz and ECFP during linkage (Fig. 3) is less of concern since this step involves the use of the Red recombinase system, which works by a gap end repair mechanism.

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