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piggyBac transposon somatic mutagenesis with an activated reporter and tracker (PB-SMART) for genetic screens in mice.

Landrette SF, Cornett JC, Ni TK, Bosenberg MW, Xu T - PLoS ONE (2011)

Bottom Line: Somatic forward genetic screens have the power to interrogate thousands of genes in a single animal.Furthermore, locating mutant clones is a prerequisite for screening and analyzing most other somatic phenotypes.We demonstrate that PB-SMART is highly mutagenic, capable of tumor induction with low copy transposons, which facilitates the mapping and identification of causative insertions.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Yale University School of Medicine, Boyer Center for Molecular Medicine, Howard Hughes Medical Institute, New Haven, Connecticut, United States of America.

ABSTRACT
Somatic forward genetic screens have the power to interrogate thousands of genes in a single animal. Retroviral and transposon mutagenesis systems in mice have been designed and deployed in somatic tissues for surveying hematopoietic and solid tumor formation. In the context of cancer, the ability to visually mark mutant cells would present tremendous advantages for identifying tumor formation, monitoring tumor growth over time, and tracking tumor infiltrations and metastases into wild-type tissues. Furthermore, locating mutant clones is a prerequisite for screening and analyzing most other somatic phenotypes. For this purpose, we developed a system using the piggyBac (PB) transposon for somatic mutagenesis with an activated reporter and tracker, called PB-SMART. The PB-SMART mouse genetic screening system can simultaneously induce somatic mutations and mark mutated cells using bioluminescence or fluorescence. The marking of mutant cells enable analyses that are not possible with current somatic mutagenesis systems, such as tracking cell proliferation and tumor growth, detecting tumor cell infiltrations, and reporting tissue mutagenesis levels by a simple ex vivo visual readout. We demonstrate that PB-SMART is highly mutagenic, capable of tumor induction with low copy transposons, which facilitates the mapping and identification of causative insertions. We further integrated a conditional transposase with the PB-SMART system, permitting tissue-specific mutagenesis with a single cross to any available Cre line. Targeting the germline, the system could also be used to conduct F1 screens. With these features, PB-SMART provides an integrated platform for individual investigators to harness the power of somatic mutagenesis and phenotypic screens to decipher the genetic basis of mammalian biology and disease.

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Tissue-specific induction of skin phenotypes by Cre-activated PB mutagenesis.(A) Luciferase signal marks clones of mutated cells in Luc-PB[mut]7;LSL-PBase;K14-Cre mice. (B) PB-induced alopecia in a PB[mut]7;LSL-PBase;K14-Cre; Ptenlox/+ mouse, (yellow dashed line) shows strong luciferase signal corresponding to hair loss (middle). Fully shaved, luciferase signal demarcates the specific region of alopecia (right). (C) Cytokeratin staining (green) of wild-type dorsal skin shows the normal thickness of the mouse epidermis (white arrowhead). DAPI (blue) marks cell nuclei. (D) Staining as in (c) from the region of alopecia reveals substantial thickening of the epidermis (white arrowhead). (E) A transposon insertion upstream of Myc was mapped to the affected region of skin. (F) Skin-specific mutagenesis in Luc-PB[mut]7;LSL-PBase;K14-Cre mice induces tumor formation and luciferase-labeled tumor cells. The white arrow corresponds to region of luciferase signal that develops into a larger tumor over time. Units, photons·s−1·cm−2·sr−1. (G) Insertion sites (wide arrows) in Gli2 were mapped in multiple skin tumors. (H) By quantitative PCR, Gli2 transcripts were upregulated in each skin tumor (BCC1, BCC2, BCC3, and BCC4) compared to WT skin. (I) The skin tumors bear resemblance to human basal cell carcinoma according to histological analysis, consistent with the activation of the Hedgehog pathway (scale bar, 50 µm).
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pone-0026650-g006: Tissue-specific induction of skin phenotypes by Cre-activated PB mutagenesis.(A) Luciferase signal marks clones of mutated cells in Luc-PB[mut]7;LSL-PBase;K14-Cre mice. (B) PB-induced alopecia in a PB[mut]7;LSL-PBase;K14-Cre; Ptenlox/+ mouse, (yellow dashed line) shows strong luciferase signal corresponding to hair loss (middle). Fully shaved, luciferase signal demarcates the specific region of alopecia (right). (C) Cytokeratin staining (green) of wild-type dorsal skin shows the normal thickness of the mouse epidermis (white arrowhead). DAPI (blue) marks cell nuclei. (D) Staining as in (c) from the region of alopecia reveals substantial thickening of the epidermis (white arrowhead). (E) A transposon insertion upstream of Myc was mapped to the affected region of skin. (F) Skin-specific mutagenesis in Luc-PB[mut]7;LSL-PBase;K14-Cre mice induces tumor formation and luciferase-labeled tumor cells. The white arrow corresponds to region of luciferase signal that develops into a larger tumor over time. Units, photons·s−1·cm−2·sr−1. (G) Insertion sites (wide arrows) in Gli2 were mapped in multiple skin tumors. (H) By quantitative PCR, Gli2 transcripts were upregulated in each skin tumor (BCC1, BCC2, BCC3, and BCC4) compared to WT skin. (I) The skin tumors bear resemblance to human basal cell carcinoma according to histological analysis, consistent with the activation of the Hedgehog pathway (scale bar, 50 µm).

Mentions: We have shown that the collective luciferase signal in tissues generated by Luc-PB[mut]7 can act as a robust reporter of mutagenesis activity. Since Luc-PB[mut]7 is also a mutator, the luciferase signal could also be used to mark the mutant cells. Given that Luc-PB[mut]7 is a low copy line and not every cell in a tissue will be luciferase positive, we reasoned that clones or patches of mutant cells will have elevated signals and could be detected through ex vivo imaging. In this sense, the luciferase reporter also behaves as a tracker for mutant cells. By monitoring ex vivo luciferase signal, we identified clonal patches in the skin and tracked the appearance of melanomas in Luc-PB[mut]7;Act-PBase;BrafCA;Tyr-CreER mice (Figures 4A and 6A; also see below). In addition, the luciferase signal enabled the identification of tumor cell infiltration into distant organs of a Luc-PB[mut]7;Act-PBase;Pten+/- mouse that developed lymphoma (Figure 4B). The enlarged thymus and lymph nodes were strongly luciferase-positive due to the dense clustering of cells that activated the luciferase reporter (Figure 4B). Strong signal was also seen emanating from a distinct portion of the liver (Figure 4B). Histology revealed that the luminescent spot was indeed infiltration of lymphocytes (Figure 4C), and we mapped identical transposon insertions from the liver infiltration and other tumor sites, indicating that the cells originated from the same clone. Thus, the Luc-PB[mut]7 line is a mutant clone tracker for monitoring clonal expansion, tumor formation, and infiltrations of cells into foreign tissues.


piggyBac transposon somatic mutagenesis with an activated reporter and tracker (PB-SMART) for genetic screens in mice.

Landrette SF, Cornett JC, Ni TK, Bosenberg MW, Xu T - PLoS ONE (2011)

Tissue-specific induction of skin phenotypes by Cre-activated PB mutagenesis.(A) Luciferase signal marks clones of mutated cells in Luc-PB[mut]7;LSL-PBase;K14-Cre mice. (B) PB-induced alopecia in a PB[mut]7;LSL-PBase;K14-Cre; Ptenlox/+ mouse, (yellow dashed line) shows strong luciferase signal corresponding to hair loss (middle). Fully shaved, luciferase signal demarcates the specific region of alopecia (right). (C) Cytokeratin staining (green) of wild-type dorsal skin shows the normal thickness of the mouse epidermis (white arrowhead). DAPI (blue) marks cell nuclei. (D) Staining as in (c) from the region of alopecia reveals substantial thickening of the epidermis (white arrowhead). (E) A transposon insertion upstream of Myc was mapped to the affected region of skin. (F) Skin-specific mutagenesis in Luc-PB[mut]7;LSL-PBase;K14-Cre mice induces tumor formation and luciferase-labeled tumor cells. The white arrow corresponds to region of luciferase signal that develops into a larger tumor over time. Units, photons·s−1·cm−2·sr−1. (G) Insertion sites (wide arrows) in Gli2 were mapped in multiple skin tumors. (H) By quantitative PCR, Gli2 transcripts were upregulated in each skin tumor (BCC1, BCC2, BCC3, and BCC4) compared to WT skin. (I) The skin tumors bear resemblance to human basal cell carcinoma according to histological analysis, consistent with the activation of the Hedgehog pathway (scale bar, 50 µm).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3198810&req=5

pone-0026650-g006: Tissue-specific induction of skin phenotypes by Cre-activated PB mutagenesis.(A) Luciferase signal marks clones of mutated cells in Luc-PB[mut]7;LSL-PBase;K14-Cre mice. (B) PB-induced alopecia in a PB[mut]7;LSL-PBase;K14-Cre; Ptenlox/+ mouse, (yellow dashed line) shows strong luciferase signal corresponding to hair loss (middle). Fully shaved, luciferase signal demarcates the specific region of alopecia (right). (C) Cytokeratin staining (green) of wild-type dorsal skin shows the normal thickness of the mouse epidermis (white arrowhead). DAPI (blue) marks cell nuclei. (D) Staining as in (c) from the region of alopecia reveals substantial thickening of the epidermis (white arrowhead). (E) A transposon insertion upstream of Myc was mapped to the affected region of skin. (F) Skin-specific mutagenesis in Luc-PB[mut]7;LSL-PBase;K14-Cre mice induces tumor formation and luciferase-labeled tumor cells. The white arrow corresponds to region of luciferase signal that develops into a larger tumor over time. Units, photons·s−1·cm−2·sr−1. (G) Insertion sites (wide arrows) in Gli2 were mapped in multiple skin tumors. (H) By quantitative PCR, Gli2 transcripts were upregulated in each skin tumor (BCC1, BCC2, BCC3, and BCC4) compared to WT skin. (I) The skin tumors bear resemblance to human basal cell carcinoma according to histological analysis, consistent with the activation of the Hedgehog pathway (scale bar, 50 µm).
Mentions: We have shown that the collective luciferase signal in tissues generated by Luc-PB[mut]7 can act as a robust reporter of mutagenesis activity. Since Luc-PB[mut]7 is also a mutator, the luciferase signal could also be used to mark the mutant cells. Given that Luc-PB[mut]7 is a low copy line and not every cell in a tissue will be luciferase positive, we reasoned that clones or patches of mutant cells will have elevated signals and could be detected through ex vivo imaging. In this sense, the luciferase reporter also behaves as a tracker for mutant cells. By monitoring ex vivo luciferase signal, we identified clonal patches in the skin and tracked the appearance of melanomas in Luc-PB[mut]7;Act-PBase;BrafCA;Tyr-CreER mice (Figures 4A and 6A; also see below). In addition, the luciferase signal enabled the identification of tumor cell infiltration into distant organs of a Luc-PB[mut]7;Act-PBase;Pten+/- mouse that developed lymphoma (Figure 4B). The enlarged thymus and lymph nodes were strongly luciferase-positive due to the dense clustering of cells that activated the luciferase reporter (Figure 4B). Strong signal was also seen emanating from a distinct portion of the liver (Figure 4B). Histology revealed that the luminescent spot was indeed infiltration of lymphocytes (Figure 4C), and we mapped identical transposon insertions from the liver infiltration and other tumor sites, indicating that the cells originated from the same clone. Thus, the Luc-PB[mut]7 line is a mutant clone tracker for monitoring clonal expansion, tumor formation, and infiltrations of cells into foreign tissues.

Bottom Line: Somatic forward genetic screens have the power to interrogate thousands of genes in a single animal.Furthermore, locating mutant clones is a prerequisite for screening and analyzing most other somatic phenotypes.We demonstrate that PB-SMART is highly mutagenic, capable of tumor induction with low copy transposons, which facilitates the mapping and identification of causative insertions.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Yale University School of Medicine, Boyer Center for Molecular Medicine, Howard Hughes Medical Institute, New Haven, Connecticut, United States of America.

ABSTRACT
Somatic forward genetic screens have the power to interrogate thousands of genes in a single animal. Retroviral and transposon mutagenesis systems in mice have been designed and deployed in somatic tissues for surveying hematopoietic and solid tumor formation. In the context of cancer, the ability to visually mark mutant cells would present tremendous advantages for identifying tumor formation, monitoring tumor growth over time, and tracking tumor infiltrations and metastases into wild-type tissues. Furthermore, locating mutant clones is a prerequisite for screening and analyzing most other somatic phenotypes. For this purpose, we developed a system using the piggyBac (PB) transposon for somatic mutagenesis with an activated reporter and tracker, called PB-SMART. The PB-SMART mouse genetic screening system can simultaneously induce somatic mutations and mark mutated cells using bioluminescence or fluorescence. The marking of mutant cells enable analyses that are not possible with current somatic mutagenesis systems, such as tracking cell proliferation and tumor growth, detecting tumor cell infiltrations, and reporting tissue mutagenesis levels by a simple ex vivo visual readout. We demonstrate that PB-SMART is highly mutagenic, capable of tumor induction with low copy transposons, which facilitates the mapping and identification of causative insertions. We further integrated a conditional transposase with the PB-SMART system, permitting tissue-specific mutagenesis with a single cross to any available Cre line. Targeting the germline, the system could also be used to conduct F1 screens. With these features, PB-SMART provides an integrated platform for individual investigators to harness the power of somatic mutagenesis and phenotypic screens to decipher the genetic basis of mammalian biology and disease.

Show MeSH
Related in: MedlinePlus